Thermostable affinity polypeptides

Abstract

To provide a method for determining an analyte in a sample.SOLUTION: A method for determining an analyte in a sample, the method comprising (a) contacting the sample with (i) a binding compound that binds to the analyte, wherein the binding compound comprises a binding agent and a first partner of an affinity couple (first affinity partner), and (ii) a second partner of the affinity couple (second affinity partner) coupled to a solid surface, an indicator and / or a second binding agent, and (b) determining the analyte based on the complex formed in step (a), wherein one of the affinity partners is a polypeptide comprising an amino acid sequence of a particular sequence or a sequence at least 50% identical thereto and the other of the affinity partners is a polypeptide comprising an amino acid sequence of a particular sequence or a sequence at least 50% identical thereto.SELECTED DRAWING: Figure 10

Description

[Technical Field]

[0001] The present invention relates to a method for determining an analyte in a sample, the method comprising: (a) contacting the sample with (i) a binding compound that binds to the analyte, the binding compound comprising a binding agent and a first partner of an affinity pair (first affinity partner), and (ii) a second partner of the affinity pair (second affinity partner) bound to a solid surface, an indicator reagent and / or a second binding agent; and (b) determining the analyte based on the complex formed in step (a), wherein one of the first affinity partner and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, and the other of the first affinity partner and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto. The present invention also relates to polypeptides, fusion polypeptides, polypeptide complexes, polynucleotides, and kits related thereto, including polypeptides comprising the amino acid sequence set forth in SEQ ID NO:1 or a sequence at least 50% identical thereto, wherein the amino acid at the position corresponding to position 77 of SEQ ID NO:1 is not histidine, and polypeptides comprising the amino acid sequence set forth in SEQ ID NO:2 or a sequence at least 50% identical thereto, wherein (i) the amino acid at the position corresponding to position 17 of SEQ ID NO:2 is not cysteine ​​and, in one embodiment, is alanine, serine, leucine, isoleucine, or glycine, and / or (ii) the polypeptide further comprises at least one functional peptide. [Background technology]

[0002] In immunoassays, for example, a widely used affinity pair for anchoring capture compounds to solid surfaces is the biotin-streptavidin affinity pair. However, especially in recent years, biotin supplementation in nutrition has become very popular, which leads to an increase in biotin content in medical samples, which may cause interference in diagnostic tests that rely on this interaction. Another widely used affinity pair is digoxigenin and anti-digoxigenin (i.e., antibodies against digoxigenin). Here too, interference has been observed due to digoxigenin-binding molecules, so-called dig-binding proteins, present in the sample. Therefore, a diagnostic test that avoids the use of previously used affinity pairs is desirable.

[0003] Group E colicins are subdivided into nine types (ColE1-ColE9), which are classified into three cytotoxic classes: membrane depolarizing agents (e.g., ColE1); nonspecific DNases (e.g., colicins E2, E7, E8, and E9); and RNases (e.g., colicins E3, E5, and E6) (reviewed in Cascales et al. (2007) Microbiol Mol Biol Rev 71(1):158). Protein-protein interactions in the colicin E9 DNase-immunity protein complex were described by Wallis et al. (1995 Biochemistry 34(42):13743). The interaction mode is based on diffusion-controlled association and femtomolar binding for the cognate complex. Garinot-Schneider et al. ((1996) J Mol Biol 260(5):731) performed random and site-directed mutagenesis on colicin E9 and identified highly conserved active site residues responsible for DNase activity. In the full-length colicin sequence, a single His 575 Ala mutation completely inactivated colicin 9 enzymatic activity. Kuhlmann et al. ((2000) J Mol Biol 301:1163) solved the 1.7 Å X-ray structure and described the structural basis for the highly specific interaction of colicin E9 from Escherichia coli (E. coli) with immunity protein 9.

[0004] Colicins have mostly been described from Enterobacteriaceae, especially E. coli To robustly utilize colicins and immunity proteins in biotechnology, colicin / immunity protein-protein interactions (PPIs) from enterobacteria have limited application due to their instability. Keeble et al. ((2006) Biochemistry 45(10):3243) showed that the melting points of the Escherichia coli (E. coli) complexes ColE2 / IM2, ColE7 / IM7, ColE8 / IM8, and ColE9 / IM9 are generally below 45°C. However, protein purification based on colicin immune affinity pairs has been proposed, for example, in WO 2017 / 100584 A1. Summary of the Invention

[0005] The technical problem underlying the present invention can be found in providing means and methods that meet the aforementioned needs and avoid the recognized problems as far as possible. The technical problem is solved by the embodiments characterized in the claims and described hereinafter.

[0006] Accordingly, the present invention provides a method for determining an analyte in a sample, comprising the steps of: (a) contacting the sample with (i) a binding compound that binds to the analyte, the binding compound comprising a binding agent and a first partner of an affinity pair (first affinity partner), and (ii) a second partner of the affinity pair (second affinity partner) bound to a solid surface, an indicator reagent, and / or a second binding agent; (b) determining the analyte based on the complex formed in step (a); Including, wherein one of the first affinity partner and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, and the other of the first affinity partner and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto. DETAILED DESCRIPTION OF THE INVENTION

[0007] Generally, terms used herein should be given their ordinary and customary meanings to those skilled in the art and should not be limited to special or customized meanings unless otherwise indicated. As used below, the terms "have," "comprise," or "include," or any grammatical variations thereof, are used non-exclusively. Thus, these terms may refer to both a situation in which no further features are present in the entity described in this context, in addition to the features introduced by these terms, and a situation in which one or more additional features are present. As an example, the expressions "A has B," "A comprises B," and "A includes B" may both refer to a situation in which no other elements exist in A other than B (i.e., a situation in which A consists solely and exclusively of B), and a situation in which one or more further elements exist in entity A other than B, such as element C, elements C and D, or further elements. Also, as will be understood by those skilled in the art, in one embodiment, the terms "comprising a" and "comprising The phrase "an" refers to "comprising one or more," i.e., is equivalent to "comprising at least one." Thus, unless otherwise indicated, a phrase referring to an item of a plurality refers in one embodiment to at least one such item, and in further embodiments to a plurality thereof; thus, for example, identifying a "cell" refers to identifying at least one cell, and in one embodiment, to identifying a multiplicity of cells.

[0008] Furthermore, when used hereinafter, the terms "preferably," "more preferably," "most preferably," "particularly," and "more particularly" are used interchangeably. The terms "e particularly," "specifically," "more specifically," or similar terms are used in conjunction with optional features without limiting further possibilities. Features introduced by these terms are therefore optional features and are not intended to constrain the scope of the claims in any way. The invention may also be implemented using alternative features, as one skilled in the art will recognize. Similarly, features introduced by "in one embodiment" or similar phrases are intended to be optional features, without any limitations on further embodiments of the invention, without any limitations on the scope of the invention, and without any limitations on the possibility of combining the feature so introduced with other optional or non-optional features.

[0009] The method specified herein is, in one embodiment, an in vitro method. The method steps may, in principle, be performed in any order deemed appropriate by a person skilled in the art, but in one embodiment, are performed in the specified order, and one or more, in one embodiment, all, of the steps may be assisted or performed by automated equipment. Furthermore, the method may include steps in addition to those explicitly mentioned above, particularly those specified below.

[0010] As used herein, the term "standard conditions," unless otherwise specified, refers to IUPAC standard ambient temperature and pressure (SATP) conditions, i.e., in one embodiment, a temperature of 25°C and an absolute pressure of 100 kPa, and in one embodiment, the standard conditions include a pH of 7. Furthermore, unless otherwise indicated, the term "about" refers to the indicated value with a technical precision generally accepted in the relevant field, in one embodiment, ±20%, in a further embodiment, ±10%, and in a further embodiment, ±5% of the indicated value. Furthermore, the term "essentially" indicates that there is no variation that affects the indicated result or use, i.e., potential variations do not cause the indicated result to deviate by more than ±20%, in a further embodiment, ±10%, and in a further embodiment, ±5%. Thus, "consisting essentially of" means including the specified components but excluding other components, excluding materials present as impurities, unavoidable materials present as a result of the process used to provide the components, and components added for purposes other than achieving the technical effect of the present invention. For example, a composition defined using the phrase "essentially consisting of" encompasses any known and acceptable additives, additives, diluents, carriers, and the like. In one embodiment, a composition consisting essentially of a set of components contains less than 5 wt. %, in a further embodiment less than 3 wt. %, in a further embodiment less than 1 wt. %, and in a further embodiment less than 0.1 wt. % of an unidentified component(s).

[0011] As used herein, the term "polypeptide" refers to a molecule consisting of a number of amino acids, typically at least 20, covalently linked to one another by peptide bonds. Molecules consisting of fewer than 20 amino acids covalently linked by peptide bonds may also be referred to as "peptides." In one embodiment, a polypeptide comprises 50-1000 amino acids, in a further embodiment 55-750 amino acids, in a further embodiment 60-500 amino acids, and in a further embodiment 65-400 amino acids. In one embodiment, the polypeptide is comprised in a fusion polypeptide and / or polypeptide complex. In one embodiment, the polypeptide is a binding compound comprising at least a binding agent and a first affinity partner, as specified herein below. In one embodiment, the polypeptide comprises at least one sequence of at least 3, in one embodiment at least 5, and in a further embodiment at least 10 contiguous amino acids not present in naturally occurring polypeptides, and in one embodiment, the additionally present sequence is an additional amino acid sequence as specified herein below. The identified polypeptide may be comprised in a fusion polypeptide comprising an additional amino acid sequence, which in one embodiment is one or more repeats of the amino acid sequence of the identified polypeptide and / or the amino acid sequence of a functional polypeptide, i.e., a polypeptide that provides at least one additional, in one embodiment, auxiliary, function. A repeat of a sequence may be an identical repeat or a repeat having at least 50% sequence identity with said polypeptide, and in one embodiment, said repeat is essentially identical to said polypeptide. A repeat may be a direct repeat, i.e., a repeat located immediately adjacent to the amino acid sequence of the polypeptide, or an indirect repeat, i.e., the sequence of the polypeptide and the sequence of the repeat are interposed by one or more further amino acid sequences, such as functional polypeptides as specified hereinabove. In one embodiment, the same applies to multiple repeats, where any two repeats may be directly or indirectly linked and may have independently selected % identity values ​​as specified hereinabove.

[0012] The functional polypeptide optionally present in the fusion polypeptide may be any polypeptide whose presence is considered desirable by those skilled in the art. In one embodiment, the functional polypeptide is selected from tag peptides, linker peptides, indicator polypeptides, and additional binding agents. The term "tag peptide" is known to those skilled in the art to refer to heterologous amino acid sequences contained in a polypeptide that provide an auxiliary function, in particular affinity tags (e.g., His6 tag, strep-tag, or GST-tag), solubilization tags (e.g., thioredoxin or poly(NANP) (SEQ ID NO: 17)), chromatography tags (e.g., His6 tag or FLAG tag), epitope tags (e.g., Myc tag, FLAG tag, or HA tag), fluorescent tags (e.g., GFP), modification tags (e.g., transglutaminase peptides or protease recognition sequences), and / or targeting tags (e.g., secretion mediators, blood-brain barrier crossing mediators, or cell-penetrating peptides). All of these tags are well known in the art. The term "linker peptide" is also known to those skilled in the art. In one embodiment, the linker peptide, which may also be referred to as a "linker," is a sequence of 2 to 25, and in one embodiment 3 to 10, amino acids, and in one embodiment, small amino acids, particularly a sequence independently selected from glycine, serine, alanine, valine, and proline. In one embodiment, the linker peptide comprises the sequence GGG, in a further embodiment the sequence GGGS (SEQ ID NO: 7), in a further embodiment the sequence (GGGS)5 (SEQ ID NO: 8), and in a further embodiment the sequence GGSSGGAGSGGG (SEQ ID NO: 9). As used herein, the term "indicator polypeptide" relates to any and all polypeptides having properties that provide the fusion polypeptide with at least one detectable physical, chemical, and / or electrochemical characteristic as specified herein below; thus, in one embodiment, the indicator polypeptide is a fluorescent tag or an enzyme such as peroxidase. The term "additional binding agent" will be understood by those skilled in the art in light of the description provided herein below. In one embodiment, the additional binding agent is not a repeat of the binding agent present in the binding compound, i.e., in one embodiment, has an amino acid sequence that has less than 50% identity to the sequence of the binding agent.In one embodiment, the further binding agent binds to the same structural, and in one embodiment, immunological, feature of the analyte, or binds to a different structural, and in one embodiment, immunological, feature of the analyte, or binds to a structural, and in one embodiment, immunological, feature of a second analyte, which is not identical to the analyte.

[0013] The phrase "the amino acid at the position corresponding to position X is not amino acid Y" is used herein in the sense that it will be understood by a person skilled in the art. Thus, the term "position corresponding to position X" in the context of a polypeptide sequence, in one embodiment, relates to a position in the polypeptide that is determined to be the position corresponding to position X by aligning the polypeptide of interest, for example, to SEQ ID NO: 1 or 2, respectively. Thus, in one embodiment, the amino acid position that corresponds to position X is the position that corresponds to position X as determined from the sequence context, but not necessarily by position number. Additionally, the phrase "is not amino acid Y" relates to any amino acid that is not amino acid Y, which in one embodiment is an alpha-amino acid, in a further embodiment an L-amino acid, and in a further embodiment an L-α-amino acid. In particular, in the context of polypeptides that are produced or producible in a host cell, amino acids that are not amino acids Y are preferred. The amino acids are proteinogenic amino acids. Thus, in one embodiment, the phrase "amino acid X is not histidine" is equivalent to the phrase "amino acid X is selected from the list consisting of alanine, valine, leucine, isoleucine, aspartic acid, glutamic acid, phenylalanine, glycine, lysine, methionine, asparagine, glutamine, proline, arginine, serine, threonine, tryptophan, tyrosine, and cysteine," and the phrase "amino acid X is not cysteine" is equivalent to the phrase "amino acid X is selected from the list consisting of alanine, valine, leucine, isoleucine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, lysine, methionine, asparagine, glutamine, proline, arginine, serine, threonine, tryptophan, and tyrosine."

[0014] The degree of identity (e.g., expressed as "% identity") between two biological sequences, in one embodiment, DNA, RNA, or amino acid sequences, can be determined by algorithms well known in the art. In one embodiment, the degree of identity is determined by comparing two optimally aligned sequences over a comparison window, where the segments of the sequences within the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to the sequences compared for optimal alignment. The percentage is calculated in one embodiment by determining the number of positions over the entire length of the polynucleotide or polypeptide where the identical nucleic acid base or amino acid residue occurs in both sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce the percentage of sequence identity. Optimal alignment of sequences for comparison can be performed using the local homology algorithm of Smith and Waterman (1981), the homology alignment algorithm of Needleman and Wunsch (1970), the similarity search method of Pearson and Lipman (1988), computer implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA, Wisconsin Genetics Software Package (Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin)), or visual inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are used in one embodiment to determine their optimal alignment, and therefore the degree of identity. In one embodiment, default values ​​of 5.00 for gap weight and 0.30 for gap weight length are used.In the context of biological sequences referred to herein, the term "at least 50% identical" includes at least 70% identity, in one embodiment at least 80% identity, in a further embodiment at least 85% identity, in a further embodiment at least 90% identity, in a further embodiment at least 95% identity, in a further embodiment at least 98% identity, and in a further embodiment at least 99% identity. Also, the term "essentially identical" refers to a percent identity value of at least 80%, in one embodiment at least 90%, in a further embodiment at least 98%, and in a further embodiment at least 99%. As will be understood, both the terms at least 50% identity and essentially identical include 100% identity. The above applies mutatis mutandis to the term "essentially complementary."

[0015] The term "fragment" of a biological macromolecule, in one embodiment a polynucleotide or polypeptide, is used herein in a broad sense to refer to any subportion, in one embodiment a subdomain, of the respective biological macromolecule comprising the indicated sequence, structure, and / or function. Thus, the term encompasses not only subportions generated by actual fragmentation of the biological macromolecule, but also subportions derived abstractly, e.g., in silico, from the respective biological macromolecule. Thus, as used herein, not only Fc or Fab fragments, but also, for example, single-chain antibodies, diabodies, and nanobodies may be referred to as fragments of antibodies. .

[0016] Unless otherwise indicated herein, the specified compounds, particularly polynucleotides and polypeptides, may be included in larger structures, and may, for example, be covalently or non-covalently linked to further sequences or further compounds, such as carrier molecules, retardants, and other excipients, particularly as specified herein above.

[0017] As indicated hereinabove, the method for determining an analyte is, in one embodiment, an in vitro method. In one embodiment, the method is a diagnostic method, i.e., a method that provides a diagnosis of a disease, or, in one embodiment, a method that does not allow the diagnosis of a disease but contributes to the establishment of a diagnosis by a physician, in one embodiment, in combination with further diagnostic methods and / or symptoms. However, the method may also be a non-diagnostic method for determining an analyte in a non-medical sample, in one embodiment, in a sample not derived from a subject, for example, in environmental analysis, industrial production analysis, e.g., quality control, etc.

[0018] As used herein, the term "determining an analyte" refers to determining at least one detectable characteristic of the analyte, which, in one embodiment, is contained in an analyte / binding compound complex. The detectable characteristic can be any characteristic considered detectable by one of skill in the art. In one embodiment, the detectable characteristic is selected from the list consisting of structural, physical, chemical, and electrochemical characteristics. Structural characteristics are all detectable characteristics conferred by the chemical structure of the analyte, particularly immunological characteristics, i.e., characteristics recognized or recognized by the binding compounds identified herein below. Physical characteristics are all characteristics detectable by physical means, e.g., optical characteristics such as absorption, transmittance, color, reflectance, fluorescence, radiation, etc. Chemical characteristics are all characteristics detectable by at least one chemical reaction, e.g., activity to produce a chemical compound, in one embodiment when a co-substrate and / or catalyst is provided; as referred to herein, chemical characteristics can be catalytic, e.g., enzymatic activity. Electrochemical characteristics include all characteristics based on a chemical reaction and detectable by electrical means, e.g., by measuring current or voltage, optionally after applying an external voltage. Electrochemical characteristics include, in particular, ECL characteristics. As will be appreciated by those skilled in the art, determining a detectable characteristic of an analyte may, in one embodiment, also include detecting the absence of said characteristic; for example, in a competitive assay, determining a characterization compound having a chemical group that confers at least one detectable characteristic contained in the characterization compound that is not present in the analyte. In one embodiment, the detectable characteristic is an immunological characteristic. As used herein, the term "immunological characteristic" refers to a structural feature of an analyte that facilitates detection of the analyte in a sample by a binding compound, particularly an antibody or derivative thereof. In one embodiment, the immunological characteristic facilitates identification and quantification of the analyte by immunological means, in further embodiments. Thus, a typical immunological characteristic is one that facilitates differentiation of the analyte from other chemical compounds in a sample.

[0019] In one embodiment, determining the analyte is establishing whether the analyte is present or absent in the sample at a concentration above the detection limit of the method, i.e., in one embodiment, the determining is qualitative. Methods for determining the detection limit are known to those skilled in the art. In a further embodiment, determining is semi-quantitative or quantitative determination of the amount or concentration of the analyte in the sample. For a semi-quantitative determination, the amount may be assigned to two or more predefined categories, for example, above or below a reference value, or low, medium, or high. For a quantitative determination, either the absolute amount or the exact amount of the analyte is determined, or the relative amount of the analyte is determined. A relative amount can be determined when the exact amount of the analyte can or cannot be determined. In this case, it can be determined whether the amount of the analyte present is increased or decreased relative to a reference sample containing the analyte in a predetermined amount. For a quantitative determination, any parameter that correlates with the amount or concentration of the analyte in the sample, or in particular a multiplication factor, can be used. Any value derived therefrom by standard mathematical and / or evaluation operations, including division, reciprocal formation, scaling, normalization, standardization, error correction, background correction, or mean or median calculation, may be determined and / or output.

[0020] The specific steps performed to determine the analyte, particularly the analyte / binding compound complex, depend on the specific assay format selected. In one embodiment, the assay is a competitive immunoassay, i.e., an immunoassay in which the analyte competes with a specified compound, such as, in one embodiment, a labeled derivative of the analyte, for binding to a binding compound, in one embodiment, the binding compound is bound to a solid surface, and the amount of specified compound bound to the binding compound is determined. In a further embodiment, the assay is a sandwich immunoassay, particularly a sandwich ECL assay, a sandwich ELISA, or a double antigen sandwich assay (DAGS). In such cases, the binding compound may be a capture compound and / or a detection compound, as specified in more detail below. In a sandwich assay, the analyte is bound to a capture compound, in one embodiment, the binding compound is bound to a solid surface, and the amount of capture compound / analyte complex is determined by binding a detection compound, as specified herein below, to the analyte / capture compound complex via the analyte. In a DAGS assay, in one embodiment, the analyte is at least bivalent, i.e., has at least two detectable features, and the binding compound and detection compound may contain the same structural feature that binds to the analyte. Thus, in one embodiment, the binder of the capture compound and the binder of the detection compound are essentially the same, and in one embodiment are identical, i.e., the capture compound and the detection compound may differ only in that the capture agent is adapted to bind to a solid surface and the detection compound comprises an indicator reagent.

[0021] As used herein, the term "analyte" relates to a chemical molecule, in one embodiment, an organic molecule, to be determined in a sample. In one embodiment, the analyte has a mass of at least 100 (corresponding to 100 atomic mass units, 1.66 x 10 -27In one embodiment, the analyte has a molecular mass of at most 2500, in a further embodiment at least 500, or in a further embodiment at least 1000. In one embodiment, the analyte is a biological molecule, and in a further embodiment, the analyte is a biological macromolecule. In a further embodiment, the analyte is a polypeptide. In one embodiment, the analyte binds to the binding compound with sufficient affinity to allow detection of the analyte / binding compound complex. In one embodiment, the analyte is troponin T and / or IGF-1 (insulin-like growth factor-1). In one embodiment, the analyte is a polypeptide antigen produced by an infectious agent, e.g., a virus or bacterium, or an antibody, e.g., an antibody produced by a subject against an antigen produced by the infectious agent. In one embodiment, the analyte is a polypeptide, and in a further embodiment, an antibody against a viral antigen, in a further embodiment, a viral polypeptide, or in a further embodiment, a viral capsid polypeptide. In one embodiment, the viral capsid polypeptide is a hepatitis virus capsid polypeptide, in one embodiment a hepatitis B virus (HB) capsid polypeptide, or in a further embodiment an HB core (HBc) antigen, or in a further embodiment a SARS-CoV-2 antigen, particularly a SARS-CoV-2 spike or nucleocapsid antigen. In one embodiment, particularly where the method employs a competitive assay format, the analyte is anti-HBc, anti-HAV (anti-hepatitis A virus), anti-HBe (anti-hepatitis B e antigen), folate, folate RBC (red blood cell), anti-TSH-R, total vitamin D, vitamin B12, thyroxine T4, FT4 (free thyroxine), thyroxine T3, FT3 (free triiodothyronine), testosterone, progesterone, digitoxin, anti-TG, anti-TPO (anti-thyroid peroxidase), DGEA, or estradiol. In one embodiment, particularly when the method uses a DAGS assay format, the analyte may be an antibody, particularly an anti-Toxoplasma IgG, anti-Rubella IgG, anti-HBs 1G, HCV (Hepatitis C Virus), CMV (Cytomegalovirus), or the like. The antibody may be a diagnostic antibody for, for example, HCV infection, syphilis, HTLV (human T-cell lymphotropic virus) infection, or Chagas (American trypanosomiasis) infection, or SARS-CoV-2 infection.

[0022] As used herein, the term "sample" refers to a sample known or suspected to contain the analyte. In one embodiment, the sample is or includes a sample of a bodily fluid, a sample from a tissue or organ, a sample of washing / rinse, or a swab or smear obtained from an external or internal surface. Bodily fluid samples include blood, plasma, serum, urine, saliva, and tears. Samples can be obtained using brushes, (cotton) swabs, spatulas, rinses / washes, punch biopsy devices, puncture of a cavity with a needle or lancet, or surgical instruments. However, in one embodiment, samples also include samples obtained by well-known techniques, including scrapings, swabs, or biopsies from the urogenital tract, perianal region, anal canal, oral cavity, upper aerodigestive tract, and epidermis. Cell-free fluids can be obtained from bodily fluids or tissues or organs by lysis techniques, such as homogenization, and / or separation techniques, such as filtration or centrifugation. It is understood that the sample may be further processed to perform this method. In particular, cells may be removed from the sample by methods and means known in the art, calcium ions may be complexed to prevent clotting, clotting may be induced, etc. Furthermore, at least one analyte may be concentrated, extracted, and / or purified from the sample by methods and means known in the art. Thus, the term sample may also relate to a preparation containing or suspected of containing at least one analyte that is diluted, concentrated, purified, and / or extracted from the sample. In one embodiment, the sample is a sample not derived from a subject, such as a water sample, a sewage sample, a food sample, etc.

[0023] As one of skill in the art will understand, the term "specific binding" and grammatical variations thereof are used herein to indicate that other compounds, typically biomolecules, present in a sample do not significantly bind to a ligand, particularly a binding compound. In one embodiment, the dissociation constant of the analyte / binding compound complex is at least 5-fold, in one embodiment at least 10-fold, and in a further embodiment at least 100-fold lower than the dissociation constant of the complex between any non-analyte compound and the binding compound in the sample.

[0024] As used herein, the term "binding compound" relates to a chemical molecule that binds to an analyte as specified herein above. The binding between the analyte and the binding compound is, in one embodiment, direct, i.e., in one embodiment, there is a direct molecular interaction between the binding compound and the analyte. However, the binding between the analyte and the binding compound may also be indirect. In one embodiment, the binding between the analyte and the binding agent is specific, in one embodiment, the binding is a group specific for a class of compounds that share structural features, e.g., specificity for IgG molecules, more preferably, said binding is analyte-specific, i.e., in one embodiment, the binding between the analyte and the binding compound is a specific binding as specified herein above. In one embodiment, the dissociation constant (K d ) can be up to 10 -8 mol / l, in further embodiments up to 10 -9 mol / l, in further embodiments up to 10 -10 mol / l, in further embodiments up to 10 -11 mol / l, in further embodiments up to 10 -12 mol / l, in an embodiment under standard conditions, and in a further embodiment under conditions specified in the examples herein.

[0025] As referred to herein, a binding compound comprises a binding agent and a first partner of an affinity pair (first affinity partner), both as specified herein. The binding agent and first affinity partner are, in one embodiment, covalently linked. In a further embodiment, at least a portion of the binding agent and first binding partner are comprised in a fusion polypeptide; for example, if the binding agent is an antibody or a fragment thereof, the heavy chain or a fragment derived therefrom may be comprised in the fusion polypeptide, and / or the light chain or a fragment derived therefrom may be comprised in the fusion polypeptide. In one embodiment, the binding agent and first binding partner in the binding compound are separated by a linker peptide, in one embodiment comprising the sequence GGSSGGAGSGGG (SEQ ID NO: 9). In one embodiment, the binding compound comprises, and in one embodiment consists of, the amino acid sequence of SEQ ID NO: 10, which is covalently linked via at least one disulfide bridge to a polypeptide comprising, and in one embodiment consisting of, SEQ ID NO: 11. In one embodiment, the binding compound comprises, and in one embodiment consists of the amino acid sequence of SEQ ID NO: 12, which is covalently linked via at least one disulfide bridge to a polypeptide comprising, and in one embodiment consisting of, SEQ ID NO: 11.

[0026] As used herein, the term "binding agent" refers, in one embodiment, to any and all compounds that specifically bind to an analyte. Binding agents are generally known to those skilled in the art. In one embodiment, the binding agent is an organic molecule, and in a further embodiment, a biological macromolecule, particularly a polypeptide as defined hereinabove. In one embodiment, the binding agent comprises one of the above-mentioned antibodies, aptamers, anticalins, designed ankyrin repeat proteins, receptors, or fragments, and in one embodiment, an antibody or a fragment thereof, having binding activity to the analyte. In one embodiment, the binding agent indirectly binds to the analyte of the present invention with sufficient affinity to allow detection of a complex comprising the analyte and the binding compound. In other words, in such cases, the binding agent is an indirect ligand. As used herein, the term "indirect binding" refers to binding in which the ligand does not directly contact the analyte but contacts a chemical molecule that binds to the analyte, and in one embodiment, specifically binds to the analyte. In one embodiment, the molecule that binds to the analyte is a molecule that directly binds to the analyte, i.e., a direct ligand. In one embodiment, the binding agent is a direct ligand of the analyte.

[0027] The term "antibody" is known to those skilled in the art. As used herein, this term includes monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two non-identical antibodies, and antibody fragments, so long as they exhibit the desired binding activity as specified elsewhere herein. In one embodiment, an antibody is a full-length antibody or an antibody fragment. The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, rather than the antibody fragments described below. These terms specifically refer to antibodies having a heavy chain including an Fc region. An "antibody fragment" includes a portion of an intact antibody, and in one embodiment, the antigen-binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, nanobodies, and multispecific antibodies formed from antibody fragments.

[0028] As used herein, the term "aptamer" refers to a macromolecule that specifically binds to its interaction partner. Aptamers can be peptide or polynucleotide aptamers and are known in principle to those skilled in the art. In one embodiment, the aptamer is a peptide aptamer, i.e., a polypeptide that specifically binds to its interaction partner and comprises 8 to 80 amino acids, in one embodiment 10 to 50 amino acids, and in a further embodiment 15 to 30 amino acids. Aptamers can be isolated from randomized libraries, e.g., randomized peptide expression libraries, in suitable host systems, such as baker's yeast. As used herein, the term "anticalin" refers to an artificial polypeptide derived from lipocalin that specifically binds to its interaction partner. Similarly, as used herein, a "designed ankyrin repeat protein" or "DARPin" refers to an artificial polypeptide that contains several ankyrin repeat motifs and specifically binds to its interaction partner. As used herein, the term "receptor" refers, in one embodiment, to a polypeptide that has the biological function of detecting the presence of a chemical compound in a biological system, e.g., a cell. , including any and all biological macromolecules in polypeptides. The receptor may be a receptor a produced by a cell or a fragment thereof that has the activity of binding to the analyte. Suitable receptors, such as estrogen receptors, folate receptors, vitamin D binding protein, and ACE-2 (angiotensin-converting enzyme), are known in the art.

[0029] The term "affinity pair" is understood by those skilled in the art. In one embodiment, the term refers to a pair of compounds, in one embodiment, polypeptides, that form a complex in aqueous solution under standard conditions. In one embodiment, the complex between the members of the affinity pair is at most 10 -9 mol / l, in one embodiment up to 10 -10 mol / l, in further embodiments up to 10 -11 mol / l, in further embodiments up to 10 -12 mol / l, in further embodiments up to 10 -13 mol / l, in further embodiments up to 10-14 The affinity pair has a dissociation constant of 0.01 mol / L. In one embodiment, the affinity pair comprises a first affinity partner and a second affinity partner, which in further embodiments are specified herein. As referred to herein, an affinity pair comprises one affinity partner comprising the amino acid sequence of at least one fragment of a colicin polypeptide from Geobacillus thermoglucosidasius (also called Parageobacillus thermoglucosidasius) or a sequence at least 50% identical thereto, and another affinity partner comprising at least one fragment of a corresponding immunity polypeptide from the same organism, or a sequence at least 50% identical thereto.

[0030] The amino acid sequences of G. thermoglucosidasius colicin polypeptides can be found in public databases, such as Genbank Accession Number 10 ... No: OUM93572.1 or UniProtKB Acc No: A0A1Y3Q1S7 and have the activity of binding to the corresponding immunity polypeptide with the affinity specified herein. Thus, in one embodiment, a fragment of a colicin polypeptide is a fragment having said activity. In one embodiment, one affinity partner comprises the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, in one embodiment a sequence at least 70% identical thereto, in a further embodiment a sequence at least 80% identical thereto, in a further embodiment a sequence at least 85% identical thereto, in a further embodiment a sequence at least 90% identical thereto, in a further embodiment a sequence at least 95% identical thereto, in a further embodiment a sequence at least 98% identical thereto, and in a further embodiment a sequence at least 99% identical thereto. Thus, in a method for determining an analyte, one affinity partner may comprise the wild-type sequence of a G. thermoglucosidasius colicin polypeptide or a fragment and / or mutein thereof. In one embodiment, the amino acid at position 77 in SEQ ID NO: 1 in the amino acid sequence of one affinity partner is not histidine, and in one embodiment is alanine, glycine, leucine, or isoleucine, and in a further embodiment is alanine. Also, in one embodiment, the amino acid at position 35 in SEQ ID NO: 1 in the amino acid sequence of one affinity partner is not cysteine, and in one embodiment is alanine, serine, leucine, isoleucine, or glycine, and in a further embodiment is alanine. Thus, in one embodiment, one affinity partner comprises, and in a further embodiment consists of, the amino acid sequence of SEQ ID NO: 3. In a further embodiment, one affinity partner further comprises at least one functional peptide, and in a further embodiment at least one linker peptide, affinity tag, and / or modification tag, as specified hereinabove. In one embodiment, one affinity partner further comprises a (His)6 or (His)8 tag, at least one GGG- or GGS-linker, and a transglutaminase peptide.In a further embodiment, one affinity partner further comprises a glycine residue preceding the sequence of SEQ ID NO: 3. In one embodiment, one affinity partner comprises, and in one embodiment consists of, the amino acid sequence of SEQ ID NO: 13.

[0031] The amino acid sequence of the G. thermoglucosidasius immunity protein is available from public databases, such as Genbank Accession No: OAO88314.1 or UniProtKB Accession No: A0A178U4V9, and has the activity of binding to the corresponding colicin polypeptide with the affinity specified herein. Thus, a fragment of the immunity polypeptide, in one embodiment, is a fragment having said activity. In one embodiment, the other affinity partner comprises the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto, in one embodiment a sequence at least 70% identical thereto, in a further embodiment a sequence at least 80% identical thereto, in a further embodiment a sequence at least 85% identical thereto, in a further embodiment a sequence at least 90% identical thereto, in a further embodiment a sequence at least 95% identical thereto, in a further embodiment a sequence at least 98% identical thereto, and in a further embodiment a sequence at least 99% identical thereto. Thus, in the method for determining an analyte, one affinity partner may comprise the wild-type sequence of a G. thermoglucosidasius immunity polypeptide. In one embodiment, in the amino acid sequence of the other affinity partner, the amino acid at position 17 of SEQ ID NO:2 is not cysteine, but in one embodiment is alanine, serine, leucine, isoleucine, or glycine, and in a further embodiment is alanine. Thus, in one embodiment, the other affinity partner comprises, and in a further embodiment consists of, the amino acid sequence of SEQ ID NO:4. In a further embodiment, the other affinity partner further comprises at least one functional peptide, and in a further embodiment, at least one linker peptide, affinity tag, and / or modification tag, as specified hereinabove. In one embodiment, one affinity partner further comprises a (His)6 or (His)8 tag, at least one GGG- or GGS-linker, and a transglutaminase peptide. In a further embodiment, the other affinity partner further comprises a glycine residue preceding the sequence of SEQ ID NO:4. In one embodiment, one affinity partner comprises, and in one embodiment consists of, the amino acid sequence of SEQ ID NO:14.

[0032] The terms "first partner of an affinity pair" and "first affinity partner," "second partner of an affinity pair" and "second affinity partner," "one affinity partner" and "the other affinity partner" are used herein only to distinguish between two partners of an affinity pair; therefore, the expression "first partner" is not related to any temporal or spatial relationship with other compounds, but is used only to distinguish it from the "second partner" of an affinity pair. The first affinity partner and the second affinity partner, and the one affinity partner and the other affinity partner, together respectively, constitute an affinity pair as specified hereinabove. As mentioned above, the first affinity partner may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, or may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto. Also, the second affinity partner may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, or may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto. In view of the above, it will be understood by those skilled in the art that if the first affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto, and vice versa. Thus, in a method for determining an analyte, both partners of the affinity pair are used. In one embodiment, the first affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto.

[0033] In one embodiment, the binding compound is a capture compound. As used herein, the term "capture compound" relates to a binding compound as specified herein above that is bound to or adapted to be bound to a solid surface.

[0034] In a further embodiment, the binding compound is a detection compound. As used herein, the term "detection compound" refers to a binding compound as identified hereinabove that is bound to an indicator reagent. In one embodiment, the detection compound is not bound to a solid surface and is not adapted to be bound to a solid surface.

[0035] As used herein, the term "indicator reagent" relates to a compound adapted to enable the detection of the presence of a molecule or complex containing said indicator reagent. Typically, an indicator reagent has a detectable property, typically an optical or / and enzymatic property.

[0036] As used herein, the term "optical property" refers to any property that can be detected by an optical instrument. Specifically, an optically detectable property can be or include at least one property selected from the group consisting of reflectance, transmittance, luminescence, scattering, fluorescence, fluorescein, diffraction, and polarization. Additional optical properties contemplated by the present invention are color, fluorescence, luminescence, or refraction. In one embodiment, the optically determinable property referred to herein refers to a property of a compound that can be optically detected, such as light absorption, luminescence, light transmissivity, or a property related thereto. It is understood that detection of an optically determinable property as used herein encompasses detection of the presence of a previously undetectable property, detection of the absence of a previously detected property, and detection of a quantitative change in the property, i.e., detection of a change in signal intensity that correlates to the degree of change in at least one optical property. It is understood that the term "optically determinable property" also relates, in one embodiment, to electrochemiluminescence, also known as electrogenerated chemiluminescence.

[0037] As used herein, the term "enzymatic property" relates to the property of an indicator reagent to generate a detectable product from a substrate by biological catalysis. Thus, enzymatic property is typically conferred by the presence of a polypeptide having said enzymatic property in said indicator reagent. Typically, the enzymatic property is at least one enzymatic activity selected from the group consisting of phosphatase activity (e.g., in alkaline phosphatase), peroxidase activity (e.g., in horseradish peroxidase), and glycosidase activity (e.g., in beta-galactosidase). Typical substrates for enzymatic activity are well known in the art. Typically, said enzymatic activity generates a product having a determinable optical property as specified hereinabove, or / and said enzymatic activity generates a product that can be determined by an electronic device.

[0038] As used herein, the term "solid surface" refers to any suitable solid surface adapted to bind a second binding partner and to be separated from a sample, e.g., by physical means. In one embodiment, the solid surface is the surface of a bead, in one embodiment, a microbead, e.g., a magnetic or paramagnetic microbead. In one embodiment, the surface is adapted to improve binding of the second binding partner, e.g., by covalently or non-covalently attaching a molecule that binds to a substructure of the second binding partner. Typical molecules that bind to a substructure of the second binding partner are, e.g., antibodies, streptavidin, complex nickel ions, etc. In a further embodiment, the solid surface binds to the second binding partner by covalent or non-covalent bonds, e.g., by hydrophobic interactions, and therefore, specific adaptation of the polypeptide for binding to the solid surface may not be required. Thus, in one embodiment, the solid surface is the surface of a multicluster plate. In one embodiment, the surface of the multicluster plate is pretreated to increase the affinity and / or capacity for binding of the capture compound. Suitable pretreatments include: This is known in the art. In one embodiment, the second binding partner is directly bound to a solid surface. However, the second binding partner may also be indirectly bound to the solid surface, for example, an antibody that specifically recognizes the second binding partner may be bound to the solid surface, or an antibody that specifically recognizes a structural feature of a fusion polypeptide comprising the second binding partner may be bound to the solid surface. In the latter case, the structural feature of the fusion polypeptide comprising the second binding partner may be, for example, a binding agent, particularly an antibody or a fragment thereof.

[0039] Step (a) of the method for determining an analyte comprises contacting the sample with (i) a first affinity partner and (ii) a second affinity partner. The term "contacting" when used in the context of the specified method is understood by those skilled in the art. In one embodiment, the term relates to physically contacting at least one compound with the sample and / or additional compounds, thereby, for example, allowing the sample to interact with the compounds. In particular, the term relates to physically contacting the first affinity partner and the second affinity partner with the sample. Depending on the assay format selected, step (a) may comprise contacting the compound with additional compounds and elements; for example, in assays that rely on the removal of uncomplexed sample components by binding of the complex to a solid surface, the mixture may further be contacted with a solid surface. In one embodiment, when the binding compound is a capture compound, said contacting may be carried out prior to step (a), e.g., by binding a second affinity partner to a solid surface before adding the sample, during step (a), e.g., by mixing beads as identified hereinabove into the mixture, or after step (a), e.g., by binding a preformed complex to the solid surface. In one embodiment, when the binding compound is a detection compound, said contacting may be carried out prior to step (a), e.g., by mixing a detection compound with the mixture before adding the sample, e.g., by mixing a detection compound with the mixture when the binding compound is a detection compound, or after step (a), e.g., by binding a preformed binding compound / analyte complex to the detection compound.

[0040] The method for determining an analyte includes a step (b) of determining the analyte based on the complex formed in step (a). The term "complex" will be understood by those skilled in the art to refer to the complex formed in step (a) related to the determination of the analyte. Thus, in one embodiment, the complex is a complex comprising at least the analyte and a binding agent. The complex can be formed at any time during the method prior to step (b), and can be formed sequentially or in one step. Sequential complex formation can be induced in one embodiment by providing a capture compound bound to a solid surface, mixing the sample, optionally washing away unbound components of the sample, adding a detection compound, followed by a further washing step. One-step complex formation can be induced, for example, by optionally mixing a capture compound bound to a solid surface, e.g., a bead, a sample, and a detection compound, and then optionally washing away all compounds not directly or indirectly bound to the solid surface, wherein at least one of the capture compound and the detection compound is a binding compound as specified herein. To determine the analyte, in one embodiment, the amount of the complex formed in step (a) is determined. In one embodiment, the liquid and solid phase components are separated from each other before determining the analyte in either or both the liquid and solid phases. In one embodiment, the amount is determined indirectly, for example, by determining the amount of a complex comprising a binding agent, in one embodiment as a capture agent, and an identifying compound. In a further embodiment, the amount is determined directly by determining the amount of a complex comprising a binding agent and an analyte, or, in one embodiment, by determining the amount of indicator reagent contained in a complex comprising a capture compound, an analyte, and a detection compound. Thus, if the binding compound is a capture compound, in one embodiment, a detection compound is added before, during, or after step (a), and if the binding compound is a detection compound, in one embodiment, a capture compound is added. In view of the above, one skilled in the art will appreciate that if the binding compound is a capture compound, the binding of the second binding partner to the solid surface occurs before the measurement in step (b), i.e., if the second affinity partner is bound to the solid surface in step (b), the amount is determined indirectly. It will be understood that the attachment of the second binding partner to a solid surface is not critical, provided that the second binding partner is attached to a solid surface.

[0041] The term "specifying compound", which may also be used as the term "specificity factor", is known in principle to those skilled in the art and relates to a compound that competes with the analyte for binding to a binding compound bound to an indicator reagent. Typically, the specifying compound is structurally similar to the analyte. Typically, the specifying compound comprises the substructure of the analyte bound by a capture compound bound to an indicator reagent. In one embodiment, the specifying compound is a compound comprising the analyte and the indicator reagent as structural elements, or the specifying compound consists of the analyte covalently bound to an indicator reagent.

[0042] As used herein, the expression "competing for binding" between two compounds to a binding compound refers to the molecular characteristics of the compound that prevent simultaneous binding to essentially the same binding site on the binding compound. Thus, typically, when an analyte and a characterization compound compete for binding to a binding compound, and the binding compound has one binding site for the analyte or characterization compound, only one molecule of the analyte or characterization compound can be bound to the binding compound at any one time. Those skilled in the art will understand that the above applies mutatis mutandis when a binding compound has two or more binding sites for an analyte or characterization compound. Those skilled in the art will know how to determine binding competition. In one embodiment, the competition for binding to a binding compound is binding to the same or essentially the same substructure of the analyte or characterization compound. In a further embodiment, when the analyte is a polypeptide, the epitope bound by the binding compound is also present in the characterization compound.

[0043] In one embodiment, the affinity pairs of the invention are used to combine two binding agents that specifically bind two non-identical analytes, e.g., to provide a capture compound that binds two non-identical analytes to a solid surface. Thus, the invention also provides a method for determining at least two analytes in a sample, comprising: (a) contacting the sample with (i) a first binding compound that binds to a first analyte, the first binding compound comprising a first binding agent and a first partner of an affinity pair (first affinity partner), (ii) a second binding compound that binds to a second analyte, the second binding compound comprising a second binding agent and a second partner of the affinity pair (second affinity partner), and (iii) a capture agent bound to a solid surface, wherein the capture agent binds to at least one of the first binding compound and the second binding compound; (b) determining the analyte based on the complex formed in step (a); Including, wherein one of the first affinity partner and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, and the other of the first affinity partner and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto.

[0044] As used herein, the term "capture agent" refers to any and all compounds that bind to a first binding agent, a second binding agent, a first affinity partner, and / or a second affinity partner and are bound to a solid surface. A capture agent, in one embodiment, is a binding agent that specifically binds to at least one of the above structures.

[0045] Advantageously, in the research underlying the present invention, it was found that colicins and immunological polypeptides from Geobacillus thermoglucosidasius form complexes that are highly stable and therefore particularly suitable for applications as affinity pairs in immunoassays, e.g., for binding the complex of interest to a solid surface. Furthermore, surprisingly, G. thermoglucosidasius (G It has been found that the B. thermoglucosidasius colicin and immunity polypeptides have high biophysical stability and are therefore suitable for use in analytical assays.

[0046] The above definitions apply mutatis mutandis below. The following further additional definitions and explanations also apply mutatis mutandis to all embodiments described herein.

[0047] The present invention further relates to a polypeptide comprising the amino acid sequence specified in SEQ ID NO: 1, or a sequence at least 50% identical thereto, wherein the amino acid at the position corresponding to position 77 of SEQ ID NO: 1 is not histidine, and in one embodiment is alanine, glycine, leucine, or isoleucine.

[0048] Thus, the polypeptide is a mutein of the G. thermoglucosidasius colicin identified hereinabove, and is also referred to as a "colicin polypeptide." As referred to herein, the designation colicin polypeptide refers to the binding activity to a cognate immunity polypeptide, but not necessarily the nuclease activity of the colicin. Thus, in one embodiment, the colicin polypeptide lacks detectable DNase activity. Furthermore, the colicin polypeptide is thermostable for at least 5 minutes at a temperature of 60°C, and in one embodiment, has a melting point of at least 60°C. Methods for determining thermostability are provided in the Examples herein. In one embodiment, in the colicin polypeptide, the amino acid at the position corresponding to position 35 of SEQ ID NO: 1 is not cysteine, and in one embodiment, is alanine, serine, leucine, isoleucine, or glycine. In a further embodiment, the colicin polypeptide further comprises at least one functional peptide, and in further embodiments, at least one linker peptide, affinity tag, and / or modification tag, as identified hereinabove. In one embodiment, the colicin polypeptide further comprises a G residue immediately preceding said amino acid sequence, and in one embodiment, an amino acid sequence MG immediately preceding said amino acid sequence, and in one embodiment, said amino acid sequence MG is the N-terminal sequence of said colicin polypeptide. In one embodiment, the colicin polypeptide further comprises a transglutaminase peptide, and in one embodiment, an amino acid sequence YRYRQ (SEQ ID NO: 15); a tag peptide, and in one embodiment, an amino acid sequence (His)6 (SEQ ID NO: 5), and in a further embodiment, (His)8 (SEQ ID NO: 6), and / or at least one linker peptide, and in one embodiment, as specified herein above. In one embodiment, the colicin polypeptide comprises the sequence of SEQ ID NO: 3, and in a further embodiment, SEQ ID NO: 16, and in a further embodiment, SEQ ID NO: 13.In one embodiment, the colicin polypeptide comprises one of the amino acid sequences above in this paragraph, or a sequence at least 75%, in one embodiment at least 85%, in a further embodiment at least 90%, in a further embodiment at least 95%, and in a further embodiment at least 98% identical thereto. In one embodiment, the colicin polypeptide is comprised in a fusion polypeptide, which in one embodiment further comprises a binding agent.

[0049] The present invention also relates to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or a sequence at least 50% identical thereto, wherein (i) the amino acid at the position corresponding to position 17 of SEQ ID NO: 2 is not cysteine ​​and, in one embodiment, is alanine, serine, leucine, isoleucine, or glycine, and / or (ii) further comprises at least one functional peptide.

[0050] Thus, the polypeptide is a mutein of the G. thermoglucosidacius immunity polypeptide as specified hereinabove, and is also referred to as an "immunity polypeptide." As referred to herein, the designation as an immunity polypeptide refers to binding activity to a cognate colicin polypeptide. Furthermore, the immunity polypeptide is thermostable at a temperature of 60°C for at least 5 minutes, and in a further embodiment, has a melting point of at least 60°C. In an embodiment, in the immunity polypeptide, the amino acid at the position corresponding to position 17 of SEQ ID NO:2 is not cysteine, and in one embodiment is alanine, serine, leucine, isoleucine, or glycine. In a further embodiment, the immunity polypeptide further comprises at least one functional peptide as identified herein above, and in a further embodiment, at least one linker peptide, affinity tag, and / or modification tag. In one embodiment, the immunity polypeptide further comprises a G residue immediately preceding the amino acid sequence, and in one embodiment, the amino acid sequence MG immediately preceding the amino acid sequence, and in one embodiment, the amino acid sequence MG is the N-terminal sequence of the immunity polypeptide. In one embodiment, the immunity polypeptide further comprises a transglutaminase peptide, in one embodiment, the amino acid sequence YRYRQ (SEQ ID NO:15); a tag peptide, in one embodiment, the amino acid sequence (HIS)6 (SEQ ID NO:5), and in a further embodiment, (His)8 (SEQ ID NO:6), and / or at least one linker peptide, in one embodiment, as identified herein above. In one embodiment, the immunity polypeptide comprises the sequence of SEQ ID NO:14, and in a further embodiment, SEQ ID NO:4. In one embodiment, the immunity polypeptide comprises one of the amino acid sequences above in this paragraph, or a sequence at least 75%, in one embodiment at least 85%, in a further embodiment at least 90%, in a further embodiment at least 95%, and in a further embodiment at least 98% identical thereto. In one embodiment, the immunity polypeptide is comprised in a fusion polypeptide, which in one embodiment further comprises a binding agent.

[0051] Therefore, the present invention also relates to a fusion polypeptide comprising a colicin polypeptide as specified herein or an immunity polypeptide as specified herein and a binding agent as specified herein above.

[0052] In one embodiment, the complex of any of the above colicin polypeptides with any of the above immunity polypeptides comprises at most 10 -9 M, in one embodiment at most 10 -10 M, in further embodiments at most 10 -11M, in further embodiments at most 10 -12 M, in further embodiments at most 10 -13 M, in further embodiments at most 10 -14 K of M D In a further embodiment, the complex of any of the above colicin polypeptides with a wild-type G. thermoglucosidasius immunity polypeptide and / or the complex of any of the above immunity polypeptides with a wild-type G. thermoglucosidasius colicin polypeptide has at most 10 -9 M, in one embodiment at most 10 -10 M, in further embodiments at most 10 -11 M, in further embodiments at most 10 -12 M, in further embodiments at most 10 -13 M, in further embodiments at most 10 -14 K of M D It has.

[0053] The present invention further relates to a polypeptide complex comprising a first partner of an affinity pair (first affinity partner) and a second partner of an affinity pair (second affinity partner), wherein (i) said first affinity partner is a colicin polypeptide as specified hereinabove and said second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto, or (ii) said first affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, and said second affinity partner is an immunity polypeptide as specified hereinabove.

[0054] The present invention also relates to polynucleotides that encode the colicin polypeptides specified herein, the immunity polypeptides specified herein and / or the fusion polypeptides specified herein.

[0055] The term "polynucleotide" is known to those skilled in the art. As used herein, this term refers to a nucleic acid comprising or consisting of a nucleic acid sequence or a nucleic acid sequence designated herein. The term includes molecules. The polynucleotides of the present invention, in one embodiment, are provided as either isolated polynucleotides (i.e., isolated from their natural context) or genetically modified forms. The polynucleotide, in one embodiment, is DNA, including cDNA, or RNA. The term encompasses single-stranded and double-stranded polynucleotides. In one embodiment, the polynucleotide is a chimeric molecule, i.e., comprises at least one nucleic acid sequence, in one embodiment, at least 20 bp, and in a further embodiment, at least 100 bp, heterologous to the remaining nucleic acid sequence. Additionally, in one embodiment, chemically modified polynucleotides are included, including naturally occurring modified polynucleotides, such as glycosylated or methylated polynucleotides, or artificially modified polynucleotides, such as biotinylated polynucleotides.

[0056] The polynucleotide encodes a polypeptide as specified elsewhere herein. In one embodiment, the polynucleotide encodes a polypeptide comprising, and in one embodiment consisting of, the amino acid sequence of SEQ ID NO: 3, 4, 13, or 14. Thus, in one embodiment, the polynucleotide comprises, and in a further embodiment, consists of, the nucleic acid sequence of SEQ ID NO: 18 or 19. It should be understood that polypeptides having the amino acid sequences detailed herein may also be encoded by more than one polynucleotide due to the degenerate genetic code. Thus, in one embodiment, the polynucleotide comprises, and in one embodiment, consists of, a nucleic acid sequence at least essentially identical to the nucleic acid sequence of SEQ ID NO: 18 or 19. The polynucleotide consists essentially of or comprises the aforementioned nucleic acid sequence. In one embodiment, the polynucleotide is an expression construct.

[0057] As used herein, the term "expression construct" relates to a polynucleotide comprising a nucleic acid sequence encoding a polypeptide as identified herein, operably linked to at least one expression control sequence that causes transcription of the coding sequence. In one embodiment, expression occurs in eukaryotic cells, prokaryotic cells, and / or isolated fractions thereof, in one embodiment, into translatable mRNA. Regulatory sequences for bacterial expression constructs, particularly promoters such as the lac promoter, are known in the art. Regulatory elements ensuring expression in eukaryotic cells, in one embodiment, mammalian cells, are also known in the art. They include, in one embodiment, regulatory sequences ensuring transcription initiation, in particular at least one promoter, and optionally a polyA signal ensuring transcription termination and transcript stabilization. In one embodiment, regulation is cell- or tissue-type specific and / or inducible, for example, by administration of a specific inducer. Preferred constitutive promoters include, for example, CMV-, SV- 40- or RSV (Rous sarcoma virus) promoter, CMV enhancer, or SV40 enhancer. Additional regulatory elements may include transcriptional and translational enhancers. In one embodiment, the enhancer is a CMV enhancer.

[0058] In one embodiment, the polynucleotide is contained in a vector. The term "vector," in one embodiment, encompasses phage, plasmid, viral, or retroviral vectors, as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes, containing other autonomously replicating polynucleotides. Furthermore, the term also relates to targeting constructs that allow random or site-specific integration of the targeting construct into genomic DNA. Such targeting constructs, in one embodiment, contain DNA of sufficient length for either homologous or heterologous recombination. In one embodiment, vectors containing the polynucleotides identified herein further comprise at least one selectable marker for propagation and / or selection in a host. Vectors can be incorporated into host cells by various techniques well known in the art. For example, plasmid vectors can be incorporated into precipitates, such as calcium phosphate precipitates or rubidium chloride precipitates, or in complexes with charged lipids, or in carbon-based classes such as fullerenes. The vector can be introduced into a host cell. Alternatively, a plasmid vector can be introduced by heat shock or electroporation techniques. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line before application to the host cell. Viral vectors can be replication-competent or replication-deficient. In the latter case, viral propagation generally occurs only in complementary hosts and / or cells. In a further embodiment, in the vector, the polynucleotide is operably linked to an expression control sequence as specified hereinabove. Preferred expression vectors are those derived from viruses such as retroviruses, particularly lentiviruses, vaccinia viruses, adeno-associated viruses, or herpes viruses, which can be used to deliver the polynucleotide or vector of the invention to a target cell population. The methods used to construct recombinant polynucleotides, expression constructs, or vectors are well known to those skilled in the art from standard textbooks.

[0059] The present invention also relates to a method for producing a colicin polypeptide as specified herein, an immunity polypeptide as specified herein and / or a fusion polypeptide as specified herein, comprising expressing a polynucleotide encoding said polypeptide or fusion polypeptide in a eukaryotic cell, or comprising expressing a polynucleotide encoding said fusion polypeptide in a eukaryotic or prokaryotic cell, in one embodiment a eukaryotic cell.

[0060] As used herein, the term "host cell" relates to any cell capable of producing the specified polypeptide or fusion polypeptide; thus, in one embodiment, the host cell is capable of receiving the expression polynucleotide and / or expression vector specified herein. In one embodiment, the host cell is a bacterial cell, and in a further embodiment, a cell of a common laboratory bacterial strain known in the art, and in one embodiment, an Escherichia strain, in particular an E. coli strain. In a further embodiment, the host cell is a eukaryotic cell, in one embodiment, a fungus, such as a yeast cell, or an animal cell. In one embodiment, the host cell is an insect cell or a mammalian cell, in particular a human, mouse, or rat cell. In a further embodiment, the host cell is a mammalian cell, such as a 293 human embryonic kidney (HEK) cell.

[0061] The method for producing a polypeptide or fusion polypeptide comprises expressing the expression polynucleotide and / or expression vector specified herein in a host cell. The term "expressing" a polynucleotide is understood by those skilled in the art. In one embodiment, the term comprises introducing the expression polypeptide and / or expression vector into a host cell, wherein the expression control sequences are selected to correspond to the host cell, i.e., in particular mammalian or mammalian viral expression control sequences are used for mammalian host cells. The above applies mutatis mutandis to other host cell types. Introduction of the expression polynucleotide and / or expression vector can be achieved by any method deemed suitable by the skilled artisan, in particular the methods specified herein above.

[0062] In one embodiment, the method comprises further steps. For example, after introduction of the expression polynucleotide and / or expression vector, the further steps may comprise incubating the host cells under conditions suitable for expression of the expression construct. Also, one or more further steps may involve recovering and / or purifying the polypeptide or polypeptides from the cells and / or culture supernatant.

[0063] The present invention also provides a method for purifying a polypeptide of interest, comprising the steps of: (A) providing a fusion polypeptide comprising the polypeptide of interest and a first partner of an affinity pair (first affinity partner); (B) contacting the fusion polypeptide of step (A) with a second partner of the affinity pair (second affinity partner) bound to a solid surface; (C) removing polypeptides that are not bound to the solid surface, thereby purifying the polypeptide of interest; Including, wherein one of the first affinity partner and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, and the other of the first affinity partner and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto.

[0064] A "polypeptide of interest" can, in principle, be any polypeptide that one skilled in the art may wish to purify. Thus, a polypeptide of interest can, in principle, be any polypeptide. In one embodiment, the polypeptide of interest comprises a binding agent as specified hereinabove.

[0065] The fusion polypeptide in step (A) can be provided in any form deemed appropriate by those skilled in the art. In one embodiment, the fusion polypeptide in step (A) is contained in a mixture containing multiple other polypeptides, particularly a host cell lysate, a host cell culture supernatant, or a partially purified preparation of the fusion polypeptide. In one embodiment, the fusion polypeptide is provided in a non-denaturing aqueous solution, particularly one of the buffers specified herein in the Examples. In one embodiment, the buffer is selected so as not to adversely affect the activity of the polypeptide of interest.

[0066] The contacting in step (B) may in particular be carried out by contacting the fusion polypeptide with a chromatographic material as a solid surface, ie as affinity chromatography.

[0067] The washing step (C) can be carried out by any method deemed appropriate by the skilled artisan depending on the purification format chosen. In one embodiment, the washing buffer is a non-denaturing aqueous solution as specified herein above.

[0068] The method may include further steps, in particular a further step (D) involving eluting the fusion polypeptide or a fragment thereof comprising the polypeptide of interest from the solid surface. The elution may be carried out by applying a denaturing buffer to the preparation obtained in step (C), thereby releasing the fusion polypeptide from the solid surface. However, the fusion polypeptide may also be designed to contain a cleavable bond between the first affinity partner and the polypeptide of interest, e.g., a protease recognition site, so that the polypeptide of interest can be released from the solid surface by adding the corresponding protease or by activating a proteolytic activity contained in the fusion polypeptide. Similarly, the polypeptide may be released by transferase activity.

[0069] The present invention also provides a kit comprising: (I) a colicin polypeptide or a polynucleotide encoding the same, and / or (II) Immunity polypeptide or polynucleotide encoding same Including, Concerning the kit included in the housing.

[0070] As used herein, the term "kit" refers to a collection of the aforementioned compounds, means, or reagents of the present invention, which may or may not be packaged together. The components of the kit may be contained in separate vials within a housing (i.e., as a kit of separate parts). The components may be individually packaged or may be provided in a single vial. As used herein, the term "housing" refers to a casing containing the specified components, which in one embodiment allows for transport, and in a further embodiment, transfer, of the compound or compounds. Furthermore, it is understood that in one embodiment, the kit is to be used to perform the methods referred to hereinabove; thus, in one embodiment, the kit is an analytical kit, in an embodiment for determining the analytes identified hereinabove, and in a further embodiment, a diagnostic kit. In such a case, in one embodiment, the colicin polypeptide and / or immunity polypeptide is included in the kit as a binding compound. In one embodiment, it is envisaged that all components are provided in a ready-to-use manner for performing at least one of the above methods. Furthermore, the kit, in one embodiment, includes instructions for performing the method. The instructions may be provided by a user manual in paper or electronic form. Furthermore, the manual may include administration and / or dosage instructions for performing the above-mentioned methods using the kit.

[0071] The present invention also relates to a device comprising a solid surface coated with (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, or (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto.

[0072] As used herein, the term "device" relates to any and all means of providing a solid surface that would be suitable for a specified coating by one skilled in the art. Thus, the device may be, for example, a multiwell plate, in one embodiment a microtiter plate, or a bead. However, other devices may also be envisioned, such as, for example, in one embodiment, a support having a flat surface, such as a slide, e.g., a glass slide; in one embodiment, a functionalized plastic surface, e.g., a dipstick or indicator strip; in one embodiment, a latex bead for use in an agglutination assay. In one embodiment, the device further comprises, in one embodiment, the other of (i) and (ii) in the complex, and / or further comprises a detection compound and / or a characterization compound.

[0073] Furthermore, the present invention relates to a system comprising a device of the present invention and means for determining the amount of detection compound and / or characterization compound present in said device.

[0074] As used herein, the term "system" refers to a system of means comprising at least the aforementioned means operatively linked to each other to enable a determination. Exemplary means for determining the amount of a complex containing an indicator and means for performing a determination are disclosed above in connection with the method of the present invention. How the means are linked in an operational manner depends on the type of means included in the device. In one embodiment, the means are included in a single device. Thus, the device may comprise (i) an analytical unit for measuring the complex and (ii) a computer unit for processing the obtained data for evaluation. Exemplary means for detection are disclosed above in connection with the embodiment of the method of the present invention. In such cases, the means are operatively linked in that the user of the system combines the results of the determination of the optically and / or electrochemically determinable properties of the indicator based on the instructions and interpretations given manually, or the instructions and interpretations are included in executable program code included in the device so that the amount or concentration of the analyte in the applied sample is output to the user as a determination result. Those skilled in the art will understand how to link the means without further ado. Exemplary systems are those that can be applied without the specific knowledge of a specialist, such as test strips or electronic devices that simply require the loading of a sample. The results may be provided as a raw data output requiring interpretation by a technician. However, in one embodiment, the output of the device is raw data that is processed, i.e., evaluated, the interpretation of which does not require a technician. Typical devices include the above-mentioned analytical units / devices (e.g., biosensors bound to ligands that specifically recognize peptides, arrays, solid supports, plasmon surface resonance devices, NMR spectrometers, mass spectrometers, etc.) or evaluation units / devices according to the methods of the present invention.

[0075] In view of the above, the following embodiments are specifically contemplated: Embodiment 1: A method for determining an analyte in a sample, comprising: (a) contacting the sample with (i) a binding compound that binds to the analyte, the binding compound comprising a binding agent and a first partner of an affinity pair (first affinity partner), and (ii) a second partner of the affinity pair (second affinity partner) bound to a solid surface, an indicator reagent, and / or a second binding agent; (b) determining the analyte based on the complex formed in step (a); Including, A method in which one of the first affinity partner and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, and the other of the first affinity partner and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto.

[0076] Embodiment 2: The method of embodiment 1, wherein the binding agent comprises one of the above antibodies, aptamers, anticalins, designed ankyrin repeat proteins, receptors, or fragments having binding activity to the analyte, and in one embodiment an antibody or fragment thereof having binding activity to the analyte.

[0077] Embodiment 3: The method of embodiment 1 or 2, wherein the binding compound is a fusion polypeptide comprising a binding agent and the first affinity partner.

[0078] Embodiment 4: The method of any one of embodiments 1 to 3, wherein the first affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto.

[0079] Embodiment 5: The method of any one of embodiments 1 to 4, wherein the first affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, and / or the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 4.

[0080] Embodiment 6: The method of any one of embodiments 1 to 5, wherein said sample is a sample from a subject, in one embodiment a tissue sample or a body fluid sample.

[0081] Embodiment 7: The method of any one of embodiments 1 to 6, wherein the sample is a plasma, serum or blood sample.

[0082] Embodiment 8: The method of any one of embodiments 1 to 7, wherein in step (b), the binding compound is a capture compound and the second affinity partner is bound to a solid surface.

[0083] Embodiment 9: The method of any one of embodiments 1 to 8, wherein in step (b), the binding compound is a capture compound and the second affinity partner is bound to a solid surface and a further capture compound.

[0084] Embodiment 10: The method further comprises contacting the sample with a detection compound, 10. The method of any one of embodiments 1-9, wherein (b) comprises determining the amount of complexes comprising said detection compound bound to said solid surface.

[0085] Embodiment 11: The method of embodiment 10, wherein the detection compound specifically binds to the analyte.

[0086] Embodiment 12: The method of embodiment 10 or 11, wherein the complex comprising the detection compound further comprises the analyte, a binding compound comprising the first affinity partner, and the second affinity partner.

[0087] Embodiment 13: The method of any one of embodiments 1 to 9, wherein the method further comprises contacting the sample with a characterization compound, and step (b) comprises determining the amount of a complex comprising the characterization compound bound to the solid surface.

[0088] Embodiment 14: The method of embodiment 13, wherein the characterization compound competes with the analyte for binding to a binding compound.

[0089] Embodiment 15: The method of embodiment 13 or 14, wherein the complex comprising the specified compound further comprises the binding agent, the first affinity partner, and the second affinity partner.

[0090] Embodiment 16: The method of any one of embodiments 1 to 15, wherein the binding compound is a detection compound and the second affinity partner is conjugated to an indicator reagent in step (b).

[0091] Embodiment 17: The method of any one of embodiments 1 to 16, wherein the binding compound is a detection compound, and in step (b) the second affinity partner is bound to an indicator and a further detection compound.

[0092] Embodiment 18: The method of any one of embodiments 1 to 17, wherein the binding compound comprises the amino acid sequence of SEQ ID NO: 10 or 12, and in one embodiment is covalently linked via at least one disulfide bridge to a polypeptide comprising SEQ ID NO: 11, and in one embodiment to a polypeptide consisting of SEQ ID NO: 11.

[0093] Embodiment 19: A polypeptide comprising an amino acid sequence as specified in SEQ ID NO: 1, or a sequence at least 50% identical thereto, wherein the amino acid at the position corresponding to position 77 of SEQ ID NO: 1 is not histidine, and in one embodiment is alanine, glycine, leucine, or isoleucine.

[0094] Embodiment 20: The polypeptide of embodiment 19, comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0095] Embodiment 21: The polypeptide of embodiment 19 or 20, wherein the amino acid at the position corresponding to position 35 of SEQ ID NO: 1 is not cysteine ​​and in one embodiment is alanine, serine, leucine, isoleucine, or glycine.

[0096] Embodiment 22: A complex of said polypeptide with a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 is at most 10 -9 M, in one embodiment at most 10 -10 M, in further embodiments at most 10 -11 M, in further embodiments at most 10 -12 M, in further embodiments at most 10 -13 M, in further embodiments at most 10 -14 K of M D 1. Embodiment 1 22. The polypeptide according to any one of 9 to 21.

[0097] Embodiment 23: A polypeptide according to any one of embodiments 19 to 22, comprising the amino acid sequence specified in SEQ ID NO: 3.

[0098] Embodiment 24: A polypeptide comprising the amino acid sequence specified in SEQ ID NO: 2 or a sequence at least 50% identical thereto, wherein (i) the amino acid at the position corresponding to position 17 of SEQ ID NO: 2 is not cysteine ​​and in one embodiment is alanine, serine, leucine, isoleucine or glycine, and / or (ii) further comprises at least one functional peptide.

[0099] Embodiment 25: A complex of said polypeptide with a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 is at most 10 -9 M, in one embodiment 10 -10 M, in further embodiments at most 10 -11 M, in further embodiments at most 10 -12 M, in further embodiments at most 10 -13 M, in further embodiments at most 10 -14 K of M D 25. The polypeptide of embodiment 24, having a value of

[0100] Embodiment 26: The polypeptide of embodiment 24 or 25, comprising the amino acid sequence specified in SEQ ID NO:4.

[0101] Embodiment 27: The polypeptide of any one of embodiments 19 to 23, wherein the polypeptide comprises an amino acid sequence identified in the preceding embodiment, or a sequence at least 75%, in one embodiment at least 85%, in a further embodiment at least 90%, in a further embodiment at least 95%, and in a further embodiment at least 98% identical thereto.

[0102] Embodiment 28: The polypeptide of any one of embodiments 19 to 27, wherein the polypeptide further comprises a G residue immediately preceding the amino acid sequence, and in one embodiment, further comprises the amino acid sequence MG immediately preceding the amino acid sequence.

[0103] Embodiment 29: The polypeptide according to any one of embodiments 19 to 28, wherein the amino acid sequence MG is the N-terminal sequence of the polypeptide.

[0104] Embodiment 30: The polypeptide of any one of embodiments 19 to 29, wherein the polypeptide further comprises a transglutaminase peptide, in one embodiment, the amino acid sequence YRYRQ (SEQ ID NO: 15).

[0105] Embodiment 31: The polypeptide of any one of embodiments 19 to 30, wherein the polypeptide further comprises a tag peptide, in one embodiment the amino acid sequence (His)6 (SEQ ID NO: 5), and in a further embodiment (His)8 (SEQ ID NO: 6).

[0106] Embodiment 32: The polypeptide according to any one of embodiments 19 to 31, wherein the polypeptide further comprises at least one linker peptide.

[0107] Embodiment 33: The polypeptide of any one of embodiments 19 to 32, wherein the polypeptide is thermostable at a temperature of 60°C for at least 5 minutes.

[0108] Embodiment 34: A fusion polypeptide comprising a polypeptide according to any one of embodiments 19 to 33 and a binding agent.

[0109] Embodiment 35: The fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 10 or 12. 35. The fusion polypeptide of embodiment 34, which in one embodiment consists of

[0110] Embodiment 36: A polypeptide complex comprising a first partner of an affinity pair (first affinity partner) and a second partner of an affinity pair (second affinity partner), in which (i) the first affinity partner is the polypeptide of embodiment 19 and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto, or (ii) the first affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, and the second affinity partner is the polypeptide of embodiment 24.

[0111] Embodiment 37: A polynucleotide encoding a polypeptide according to any one of embodiments 19 to 33 and / or a fusion polypeptide according to embodiment 34 or 35.

[0112] Embodiment 38: A method for producing a polypeptide according to any one of embodiments 19 to 33 and / or a fusion polypeptide according to embodiment 34 or 35, comprising expressing a polynucleotide encoding said polypeptide or fusion polypeptide in a eukaryotic host cell, or expressing a polynucleotide encoding said fusion polypeptide in a eukaryotic or prokaryotic host cell, in one embodiment a eukaryotic host cell.

[0113] Embodiment 39: The method of embodiment 38, wherein the eukaryotic host cell is a mammalian host cell, in one embodiment a human host cell, and in a further embodiment a 293 human embryonic kidney (HEK) cell.

[0114] Embodiment 40: The method of embodiment 38 or 39, wherein the polynucleotide encoding the polypeptide is the polynucleotide of embodiment 37.

[0115] Embodiment 41: A method for purifying a polypeptide of interest, comprising: (A) providing a fusion polypeptide comprising the polypeptide of interest and a first partner of an affinity pair (first affinity partner); (B) contacting the fusion polypeptide of step (A) with a second partner of the affinity pair (second affinity partner) bound to the solid surface; (C) removing polypeptides that are not bound to the solid surface, thereby purifying the polypeptide of interest; Including, A method in which one of the first affinity partner and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, and the other of the first affinity partner and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto.

[0116] Embodiment 42: A kit comprising: (I) a polypeptide (first affinity partner) according to any one of embodiments 19 to 23 and 27 to 33 or a polynucleotide encoding the same, and / or (II) A polypeptide (second affinity partner) according to any one of embodiments 24 to 33 or a polynucleotide encoding the same. Including, Housing included in the kit.

[0117] Embodiment 43: (i) the amino acid sequence of SEQ ID NO: 1 or at least 50% identical thereto or (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto.

[0118] Embodiment 44: A device according to embodiment 43, wherein said device is a multiwell plate, in one embodiment a microtiter plate, or a bead.

[0119] Embodiment 45: The device of embodiment 43 or 44, further comprising the other of (i) and (ii), in one embodiment in a composite.

[0120] Embodiment 46: A device according to any one of embodiments 43 to 45, further comprising a detection compound and / or a characterization compound.

[0121] Embodiment 47: A system comprising a device according to any one of embodiments 43 to 46 and means for determining the amount of detection compound and / or characterization compound present in said device.

[0122] Embodiment 48. A method for determining at least two analytes in a sample, said method comprising: (a) contacting the sample with (i) a first binding compound that binds to a first analyte, the first binding compound comprising a first binding agent and a first partner of an affinity pair (first affinity partner), (ii) a second binding compound that binds to a second analyte, the second binding compound comprising a second binding agent and a second partner of the affinity pair (second affinity partner), and (iii) a capture agent bound to a solid surface, wherein the capture agent binds to at least one of the first binding compound and the second binding compound; (b) determining the analyte based on the complex formed in step (a); Including, A method in which one of the first affinity partner and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, and the other of the first affinity partner and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto.

[0123] Embodiment 49: The method of embodiment 48, wherein the capture agent binds to at least one of the first binding agent and the second binding agent.

[0124] Embodiment 50: The method of embodiment 48 or 49, wherein the capture agent binds to at least one of the first affinity partner and the second affinity partner.

[0125] All references cited herein are hereby incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned herein. [Brief explanation of the drawings]

[0126] [Figure 1] Ni-NTA purified Fab <tnt>- Western blot analysis of GthColT protein; 1: size marker, 2: flow-through, 3: wash fraction, 4-5: elution fraction. [Figure 2] Ni-NTA purified Fab <tnt>- Western blot analysis of GthIM protein; 1: flow-through, 2-4: wash fractions, 4-6: elution fractions. [Figure 3] Anti-His Western blot showing purification of GthCol M1: Novex™ Sharp prestained protein standard. M2: MagicMark™ XP Western protein standard. Lane 1: Reference solution. Lane 2: Flow-through. Lanes 3-4 represent the eluted peak fractions. The arrow indicates the 15.7 kDa GthCol. [Figure 4] SDS-PAGE showing the purification of GthCol. M: Novex™ Sharp prestained protein standard. Lane 1: Reference solution. Lane 2: Flow-through. Lanes 3-4 represent the eluted peak fractions. The arrow indicates the 15.7 kDa GthCol. [Figure 5] SDS-PAGE showing SEC purification of GthIM. M: Novex™ Sharp prestained protein standard. Lane 1: Reference solution. Lanes 2-10: Elution fractions. The arrow indicates the 10.5 kDa GthIM. [Figure 6] Anti-His Western blot showing SEC purification of GthIm. M1: Novex™ Sharp prestained protein standard. M2: MagicMark™ XP Western protein standard. See lane 1. Lanes 2-10: elution fractions. Arrow indicates 10.5 kDa bound protein. [Figure 7] DSC analysis of GthIM. N=2. Tm 79°C. [Figure 8] DSC analysis of GthCol. N=2. Tm 63°C. [Figure 9] DSC analysis of GthIM / GthCol complex 1:3 in 150 mM KCl. N=2. Tm 96°C. [Figure 10] Schematic Biacore assay to investigate the interactions and cross-reactivities between GthCol, GthIM, E. coli IM-7, and ColE-7. [Figure 11] Biacore sensorgrams comparing E. coli and Geobacillus thermoglucosidasius colicin / immunity protein kinetic signatures, GthIM / GthCol interactions with superior kinetics. [Figure 12] Schematic Biacore assay. The target is surface-displayed Fab. <tnt>-The purpose of this study was to investigate the accessibility of TnT and GthCol to GthIM. [Figure 13] Antibody Fab Fragment Fab <tnt>- Biacore sensorgram showing the high affinity interaction of GthIM with GthCol at 37°C. The Fab fragment-associated GthIM binding site is fully accessible. [Figure 14] Fab <tnt>-Biacore sensorgram showing GthIM / TnT 37°C kinetics. [Figure 15] Schematic Biacore assay. Surface-displayed Fab <tnt>-GthIM, TnT and IgG antibody clones <tnt>Testing for ternary complex formation between M-11-7-variants (see WO 2021 / 028309 A1). [Figure 16] Surface displayed <tnt>-GthIM complex formation, binding to a mixture of GthCol and TnT (1), followed by (2) sandwich-forming IgG <tnt>-M-11-7 variant, and (3) the final complex dissociation phase. Gray arrows indicate the start and stop of the infusion. [Figure 17] Schematic Biacore assay. Surface-displayed Fab <tnt>-GthIM, TnT and IgG antibody clones <tnt>Testing for ternary complex formation between M-11-7-variants. [Figure 18] A.)Fab <igf-1>-GthIM and B.)Fab <igf-1>-IgG displayed on the surface of GthCol, A and B <tnt>-11-7-GthIM C.)Fab <igf-1>-GthIM and D.)Fab <igf-1>- Surface-displayed IgG of GthCol, C and D, respectively <tnt>An exemplary Biacore sensorgram showing a 30 nM injection of -11-7-GthCol. A positive response signal was obtained only when the GthCol and GthIM domain fusions formed a high affinity complex. [Figure 19] Binds to their antigens TnT and IGF-1 <tnt>Bispecific antibody complexes and <igf-1>Biacore sensorgrams showing GthCol / GthIm fused to a bispecific antibody complex. A) IgG <tnt>-11-7-GthCol / Fab <igf-1>-IGF-1 binding to the GthIM complex; B) IgG <tnt>-11-7-GthCol / Fab <igf-1>- Binding of TnT to the GthIM complex; C) IgG <tnt>-11-7-GthIM / Fab <igf1>-IGF-1 binding to the GthCol complex; D) IgG <tnt>-11-7-GthIM / Fab <igf-1>-Binding of TnT to the GthCol complex. [Figure 20] Model of GthCol / GthIM trimeric bispecific antibody. One IgG <tnt>-11-7-GthIM (ribbon) is made up of two Fab <igf1>-GthCol (transparent surface view) A trimeric antibody with dual specificity for IGF-1 and TnT is formed via the GthCol / GthIM binding module.

[0127] The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention in any way. [Example]

[0128] Example 1: Preparation of GthCol and GthIm Example 1.1 Construction of expression plasmids The DNA sequences of the Geobacillus thermoglucosidasius C-terminal colicin DNase domain and its cognate immunity protein were identified by 3-DM data mining and obtained from UniProt (A0A1Y3Q1S7 and A0A178U4V9). The sequences of GthCol and GthIm were engineered as follows: an additional glycine residue was inserted as the first amino acid after the initiating methionine into the polypeptide chain. For further purification and conjugation, a transglutaminase substrate tag (Q-tag) and an octahistidine tag were added to the C-terminus of the protein via a glycine / serine linker. For GthCol, the cysteine ​​at position 36 was exchanged with an alanine (C36A). To inactivate the DNase activity, the histidine at position 78 was exchanged with an alanine (H78A). For GthIm, the cysteine ​​at position 18 was substituted with alanine (C18A).

[0129] All gene synthesis was designed for the pQE-80 L plasmid (Qiagen) and ordered from GeneArt. The E. coli expression vector was generated by molecular cloning of the GthCol and GthIm fragments into the plasmid pQE80-Kan using the restriction enzymes EcoRI-HF and HindIII-HF (NEB). Transformation was performed using chemically competent E. coli XL1-Blue cells (Agilent) according to the manufacturer's protocol. To select successfully transformed cells, cells were spread onto lysogeny broth (LB) agar plates containing 50 μg / mL kanamycin. The plates were incubated overnight at 37°C. A 5 mL LB culture was inoculated with a single colony and grown overnight at 37°C. Plasmid DNA was isolated according to the Qiagen Miniprep kit and finally transformed into the E. coli expression strain BL21 (Agilent). Glycerol stocks for inoculation of subsequent cultures were prepared by adding 20% ​​glycerol to the starter culture and immediately flash-freezing in liquid nitrogen.

[0130] Example 1.2: Protein production in E. coli Use 250 mL of the overnight culture to inoculate a 1.5 L culture containing 50 μg / mL kanamycin and grow to an OD of approximately 0.8 with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG). 600 The lac promoter was induced with . Expression was allowed to continue for 4 hours at 37°C. Cells were harvested by centrifugation at 6,000 g for 20 minutes at 4°C. Cell extracts were prepared by French press lysis at 1.5 kbar in 20 mM Tris, 0.5 M NaCl, 5% glycerol, 20 mM imidazole, pH 8. Lysates were collected at 4°C. The supernatant was clarified by centrifugation at 12,000 rpm for 30 min at 25°C. Protease inhibitors (Complete Protease Inhibitor Cocktail, Roche) and DNase (DNase I, Roche) were added to prevent proteolysis and reduce the viscosity of the lysate. The supernatant was sterile filtered (0.2 μm) and purified by AKTA™ Pure chromatography. Equilibrate the Ni system using 2+- The mixture was applied to an NTA column (HisTrapHP, GE Healthcare). Nonspecific and unbound proteins were removed by alternating washings with 20 mM Tris, 1 M NaCl, 5% glycerol, 50 mM imidazole, pH 8 and 20 mM Tris, 5% glycerol, 50 mM imidazole, pH 8. Finally, the protein was eluted with 5 CV (column volumes) of 20 mM Tris, 0.5 M NaCl, 5% glycerol, 250 mM imidazole, pH 8. Protein-containing fractions were pooled and further purified by size exclusion chromatography (HiLoad 60 / 600 Superdex 75 pg, GE Healthcare). Prior to application, the sample was concentrated to a final volume of 1 mL or less using an ultracentrifugal column (Amicon™ Ultracentrifugal Filter Device, Merck). The column was equilibrated and the protein was eluted with 1.5 CV of 20 mM Tris, 150 mM NaCl, pH 8 at a flow rate of 1 mL / min. The desired peak fractions were pooled, concentrated, and frozen in nitrogen until further use. Protein purity was confirmed by SDS-PAGE and Western blot.

[0131] In another experiment, protein expression was carried out in a 6L laboratory-scale fermenter. The culture medium was fermentation medium, and the pO2 value was adjusted to 50%-30% by feeding 75% glycerol at 30°C. The culture was continued for 22.5 hours until the OD600 reached 46. The cells were harvested and resuspended in a pH 7.4 buffer (20 mM Na2HPO4, 500 mM NaCl, 5% glycerol, and 20 mM imidazole). Cell disruption was carried out using a high-pressure homogenizer at 1500 bar. Prior to enzyme purification, the cell extract was pretreated with 1-2% Polymin-G20.

[0132] Example 1.3: Cell culture production of antibody fusion proteins Sequence analysis of the variable heavy and light chains of monoclonal mouse IgG was performed by RACE PCR after mRNA isolation using the 5' / 3' RACE kit 2nd Gen (Roche Diagnostics GmbH) and PCR primers as described previously (Doenecke et al. (1997), Leukemia 11:1787). PCR amplicons were purified and directly sequenced using the same primers.

[0133] For the monoclonal antibody anti-TnT M-11-7 (see WO 2021 / 028309 A1), the mouse variable coding regions were fused to human immunoglobulin gamma-1 heavy chain (sw_hum:IGG1_HUMAN) and immunoglobulin kappa constant (sw_hum:IGKC_HUMAN) coding regions to obtain a chimeric mouse / human antibody.

[0134] Subsequently, either the ColA0A (Geobacillus thermoglucosidasius colicin, A0A1Y3Q1S7) C36A, H78A mutant or ColA0A_ImP (Bacillaceae bacteriocin immunity protein, A0A178U4V9) C18A mutant protein fragment was fused to the C-terminus of all three heavy chain fragments.

[0135] Fab <igf-1>Fab (see, e.g., WO 2012 / 150321) and Fab <tnt>For secretion of the M-11-7 fusion protein, a modified serum albumin leader sequence was attached to the N-terminus. Gene synthesis, including codon optimization, was performed using Geneart (Thermo Fisher Scientific). The expression construct was then inserted via SalI / BamHI into the eukaryotic expression vector pM1MT (Roche). The clone was subcloned at NIH Molecular Biochemicals.

[0136] After gene synthesis with Geneart, <tnt>The -M-11-7 expression construct was subcloned via BsmI in frame into the variable heavy chain mouse leader sequence characterized by an intron as previously described (Norderhaug et al. (1997), J. Immunol. Methods. 204:77-87).

[0137] Eukaryotic expression was under the control of the human cytomegalovirus immediate-early enhancer / promoter region followed by the bovine growth hormone polyadenylation signal.

[0138] For transient gene expression in human embryonic kidney (HEK) 293 cells, the FreeStyle™ 293-F Expression System (Thermo Fischer Scientific) was used: approximately 2 × 10 6 Cells / ml of culture were transfected with the respective LC / HC expression plasmid pairs at a concentration of 0.5–1 mg / l cell culture complexed with 293-Free™ transfection reagent (Merck Millipore) according to the manufacturer's guidelines. Three hours after transfection, the histone deacetylase inhibitor valproic acid was added to 4 mM to promote protein production (Backliwal et al. (2008) Biotechnology and Bioengineering 101:182). Cultures were supplemented daily with a 6% (v / v) soybean peptone hydrolysate-based diet. Seven days after transfection, culture supernatants were harvested, cleared from cell debris by centrifugation, and stored at -80°C for purification.

[0139] Example 1.4: Purification of antibody fusion proteins The binding protein GthCol (16 kDa) or GthIm (13 kDa) was attached to Fab via a flexible amino acid linker. <tnt>-M-11-7A102W-Q-H6(Fab <tnt>-GthCol heavy chain, Fab <tnt>-GthIM and Fab <igf-1>The Fab fragment fusion proteins were expressed and then fused to the Ni fragments (SEQ ID NOs: 10 and 12, respectively). 2+- NTA was purified from HEK 293 cell supernatant. The supernatant was sterile filtered (0.2 μm) and the medium was replaced with 20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4. AKTA™ Pure chromatography was performed. Equilibrated Ni using the Fee system 2+- NTA column (HisTrapHP, GE The sample was applied to a column (Division of Biological Sciences, University of California, San Diego, CA). Nonspecific and unbound proteins were removed by washing the column with 5 CV of sample buffer. Finally, the protein was eluted with 5 CV of 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4. The desired peak fractions were pooled, and the buffer was exchanged into 50 mM potassium phosphate, 150 mM KCl, pH 7.4 (Amicon® Ultracentrifugal Filter Device, Merck). The protein solution was frozen in nitrogen until further use. Protein purity was confirmed by SDS-PAGE and Western blot.

[0140] Example 1.5: SDS-PAGE and Western Blot Analysis SDS-PAGE analysis was performed under reducing conditions using NuPAGE® Bis-Tris 4-12% gels (Thermo Fisher). Immunoblotting on nitrocellulose membranes (iBlot™ Gel Transfer Stack Nitrocellulose, midi / mini, Thermo Fisher) was performed according to the manufacturer's instructions using anti-His-peroxidase (Roche) for detection. Blots were visualized using Lumi-LightPLUS Western Blotting Substrate (Roche). The results are shown in Figures 1-6.

[0141] Example 1.6: Determining Protein Stability by Differential Screening Calorimetry (DSC) Protein melting temperatures (Tm) were measured using a Microcal-VP-DSC (Malvern DSC measurements were performed by Panalytical. The DSC approach was performed using a heating rate of 1.5 °C / min from 20 °C to 120 °C, with duplicates of each sample at a concentration of 1 mg / mL in 50 mM KH2PO4, 150 mM KCl, pH 7.4. Data analysis was performed using Origin (peak analysis of the second derivative of the Cp raw data).

[0142] Example 1.7: Determining Protein Stability by Differential Scanning Fluorometry (DSF) Thermal unfolding experiments were performed using Prometheus NT.Plex. Fluorescence intensity and fluorescence maximum depend on the local tryptophan environment in each protein's structure. Therefore, the ratio of fluorescence intensities at 350 nm and 330 nm is suitable for detecting changes in protein structure, e.g., due to protein unfolding. Samples were measured in triplicate. 2 mg / ml E. coli Col-E7, 2.7 mg / ml E. coli IM-7, 0.25 mg / ml GthCol, and 0.7 mg / ml GthIM were measured in 50 mM KH2PO4, 150 mM KCl, pH 7.4. The temperature was increased from 20°C to 90°C. The fluorescence ratio 350 nm / 330 nm was determined.

[0143] The results of the DSF and DSC measurements are shown in FIGS. [Table 1]

[0144] DSC melting experiments showed that the Tm values ​​of GthIM, GthCol, and especially the GthIM / GthCol complex were sufficiently stable for robust biotechnological applications even under high-salt conditions of 150 mM KCl.

[0145] Example 2: Interaction analysis of E. coli IM7 / ColE, GthIM / GthCol, IM7 / GthCol and GthIM / ColE, and multispecific constructs Example 2.1: BIAcore experiments Kinetic studies of E. coli Im7 / ColE-7 and Geobacillus thermoglucosidasius GthIM / GthCol were performed at 37°C using a BIAcore T200 instrument (formerly GE Healthcare). The objective was to investigate the affinity and specificity between GthIM / GthCol and IM7 / ColE-7. Enzymatically monobiotinylated IM-7 (15 kDa) and monobiotinylated GthIM (13 kDa) were immobilized on a streptavidin sensor surface, and ColE-7 (18 kDa) and GthCol (16 kDa) were used as analytes in solution. HBS-ET (10 mM HEPES, 150 mM NaCl, pH 7.4, 3 mM EDTA, 0.05% (w / v) Tween 20) was used as the running and sample buffers.

[0146] Install a Biacore Streptavidin Series S sensor (catalog no. BR-1005-31; lot no. 10271085) into the instrument and use the instrument according to the manufacturer's instructions. The samples were pretreated as described. Biotinylated IM-7 was injected onto flow cell 2 using a concentration of c = 5 nM with a contact time of 100 s and a flow rate of 10 μL / min. A ligand density of 204 resonance units (RU) was obtained for Im7. Free SA binding sites were blocked following Im7 immobilization: amino-PEO-biotin was injected onto flow cells 1 and 2 using a concentration of c = 50 μM with a contact time of 3 min and a flow rate of 30 μL / min, with flow cell 1 used as a reference for kinetic studies. In a separate experiment, biotinylated GthIM (13 kDa) was immobilized onto a second SA sensor as described above, yielding a ligand density of 139 RU on flow cell 3 for GthIM.

[0147] Kinetic characterization was performed using single-cycle kinetics. Five consecutive concentrations of ColE-7 (18 kDa) or GthCol (16 kDa) ranging from 1.1 nM to 90 nM were injected at 60 μL / min with an association time of 5 min. The dissociation phase was monitored for 1 h after the final injection. Buffer injection was used as the reference. Data were evaluated using double referencing to correct for bulk effects and systematic noise. The association rate constant k a [M -1 s -1 ] and the dissociation rate constant k d [s -1 The complex half-life (min) was calculated using the Langmuir 1:1 fitting model according to the BIAcore™ T200 Evaluation SW V 3.2 from GE Healthcare. d was calculated according to

[0148] The resulting affinity is calculated using the formula: K D =k d / k a Calculated using [M]. The molar ratio and binding stoichiometry are calculated using the formula: MW(Im) / MW(Col)*R max The calculation was performed using exp.(Col) / ligand density (Tm). The outline is shown in Figure 10, and the results are shown in Figure 11 and Table 2.

[0149] The binding signature of GthIm / Gth is characterized by the association rate constant k a >5.0E+07M -1 s -1 The complex dissociation was measured using the instrument specification k d <1.0E-05s -1 GthIM saturation is achieved at 3 nM GthCol. The GthIM / GthCol complex is very stable at 37°C. The apparent affinity K D <2.0E-13M -1 is clearly outside the instrument specifications. The molar ratio of 1.1 indicates a 1:1 binding stoichiometry. The binding signature of GthIM / ColE7 is characterized by an association rate constant k a >1.0E+07M -1 s -1 and the complex exhibits a fast on / off profile with a half-life time t / 2-diss of <1 min. D = 13 nM. GthIM interacts nonspecifically with ColE7 with approximately 65,000-fold lower affinity when compared to GthIM / GthCol kinetics.

[0150] The IM7 / ColE7 kinetic profile exhibits an association rate constant, k a 1.8E+06 M -1 s -1 shows rapid complex formation until saturation occurs. d <1.0E-05s -1 ) results in a complex half-life t / 2-diss>1155. Affinity K D =5.7E-12M -1 The constant obtained is outside the instrument specifications. The molar ratio of 1.2 is 1:1.

[0151] The binding stoichiometry is shown. The GthIm / GthCol interaction is at least 30 times stronger than the IM7 / ColE7 interaction. The binding signature of IM7 / GthCol is characterized by an association rate constant k a 1.2E+06 M -1 s -1 The complex formation rate is fast at 100 kJ / s. d 9.2E-05 s -1 ) has a complex half-life of t / 2-diss 126 min and affinity K D = 74 pM. [Table 2]

[0152] Example 2.2: Fab <tnt>-Analysis of the interaction of GthCol with GthIM and TnT In this experiment, the aim was to identify Fab fragments with their cognate antigen TnT (37 kDa) and GthIM (13 kDa) binding domains. <tnt>The objective of this study was to investigate the antigen accessibility of -GthCol (64 kDa).

[0153] A Biacore CM5 Series S sensor (catalog no. BR29-1496-03, lot no. 10281824) was installed in the instrument.<M-IgG F(ab’)2> Fab R (Jackson Immuno Research, Catalog No. #315-005-047, Lot #107797) was immobilized as a capture system according to the manufacturer's instructions with a maximum of 9829 RU. The system and sample buffer was HBS-EP (10 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.05% (w / v) P20, pH 7.4). Flow cells 2, 3, and 4 were used for interaction measurements, and flow cell 1 was used as a reference. <tnt>-GthIM (59 kDa) was diluted in steps of 10 nM, 1 nM, and 0.3 nM and injected at 5 μL / min for 3 min. Fab in resonance units [RU] <tnt>-GthIM capture level (CL) was monitored.

[0154] Increasing concentrations of GthCol (16 kDa) from 1.1 nM to 90 nM were displayed on the surface in duplicate at 60 μL / min. <tnt>-Fab-GthIM. The association phase was monitored for 5 min and the dissociation phase was monitored for 10 min and 1 h, respectively, at 3.3 nM.

[0155] The TnT (37 kDa, Roche in-house) concentration was increased from 0.4 nM to 30 nM, and 10 nM copies of the surface-displayed Fab were injected at 60 μL / min. <tnt>-GthIM (59 kDa). The association phase was monitored for 5 min, and the dissociation phase was monitored for 1 h and 10 min, respectively, for a 10 nM concentration. After each cycle, Fab <tnt>-GthIM was regenerated from the capture system by injecting 100 mM HCl at a flow rate of 20 μL / min for 3 min. A schematic is shown in Figure 12 and the results are shown in Figure 13.

[0156] Fab <tnt>-GthIm binds to the analyte GthCol with rapid complex formation and saturation. No dissociation was observed within 1 hour. The association rate constant was k a 9.9E+06 The apparent dissociation (k d <1.0E-05s-1) was outside the instrument specifications. D <1.0E-11 M. A molar ratio of 1.0 indicates a 1:1 binding stoichiometry.

[0157] Example 2.3: Fab <tnt>-GthIM-binding troponin T (TnT) Fab <tnt>-GthIM is the association rate constant k a 7.6E+05 M -1 s - 1 Binds to TnT with rapid complex formation at 100 kJ / s. The dissociation rate constant is k d 9.2E-05 s -1 yields a complex half-life t / 2-diss of 125 min. The resulting affinity K D is 121 pM. Molar ratio = 0.7 indicates a 1:1 binding stoichiometry. The results are shown in Figure 14 and Table 3. [Table 3]

[0158] Example 2.4: Tertiary immune complex formation on TnT In this experiment, the aim was to investigate the accessibility of each binding partner.

[0159] The capture system was used as described in the previous experiment. <tnt>-GthIM (59 kDa) was captured at 5 μL / min for 2 min. Free capture system binding sites were blocked with 1 μM mouse IgG Fab' fragment (37 kDa) that does not recognize TnT or 1 μM mouse IgG Fab' fragment (16 kDa) that does not recognize GthCol.

[0160] Using the double injection function, two consecutive injections were performed: in the first, a mixture of TnT (37 kDa) and Col (16 kDa) at a concentration of 30 nM each was injected onto the surface-displayed Fab. <tnt>The second injection was 50 nM of IgG antibody at a flow rate of 20 μL / min for 5 minutes. <tnt>The M-11-7 variant (160 kDa) was injected at 20 μl / min for 5 min. Regeneration after each cycle was performed as described. A schematic is shown in Figure 15 and the results are shown in Figure 16.

[0161] The GthCol and TnT binding sites are accessible. Further experiments (data not shown) demonstrated that colicin and TnT bind independently of each other, as demonstrated by permuting the injection order. When presented as a mixture, both targets bind, resulting in the expected additive response. IgG <tnt>-M-11-7 variant is Fab <tnt>- Recognizes a different TnT epitope from GthIM. The GthIM-binding domain does not interfere with antigen sandwich formation.

[0162] Example 2.5: GthCol / GthIM-mediated conjugation of bispecific antibody constructs This experiment aimed to investigate the ability to form an avid bispecific antibody format.

[0163] Kinetic studies were performed at 37°C using a GE Healthcare BIAcore T200 instrument. mAb IgG <tnt>rH-M-11-7-GthCol(173 kDa) and IgG mAb <tnt>rH-M-11-7-GthIM (163 kDa) was captured on the surface of a CM5 series S sensor, <igf1>-Fab-GthCol (64 kDa) or Fab <igf1>-GthIM (59 kDa) was used as the analyte in solution. System and sample buffer HBS-ET (10 mM HEPES, 15 0 mM NaCl, pH 7.4, 3 mM EDTA, 0.05% (w / v) Tween 20). A CM5 Series S sensor (catalog number BR29-1496-03, lot number 10286865) was installed in the instrument. Monoclonal mAb <h-fc-pan>M-R10Z8E9 (in-house Roche, lot number 2) was immobilized at 14509 RU on the sensor surface as a capture system. The antibody was amine coupled using EDC / NHS chemistry according to the manufacturer's instructions.

[0164] Flow cells 2, 3, and 4 were used for the measurements, and flow cell 1 was used as a reference. 0.5 nM of each antibody was captured on a different flow cell at 5 μL / min for 2 min. pAb<->H-IgG was injected into flow cell 1 as a reference. The capture level (CL) in resonance units [RU] was monitored. 30 nM analyte <igf1>-Fab-GthCol or <igf1>-Fab-GthIM, a surface-displayed mAb <tnt>rH-M-11-7-GthCol-IgG and mAb <tnt>rH-M-11-7-GthIM-IgG was injected at 30 μL / min. To obtain kinetics, a series of analyte concentrations from 0.04 nM to 30 nM was injected at 30 μL / min onto the surface-displayed antibody in duplicate for 1.1 nM. The association phase was monitored for 5 min, and the dissociation phase was monitored for 10 min. Regeneration from the capture system was performed with 10 mM NaOH for 15 s at a flow rate of 20 μL / min, followed by a 2-min injection of 10 mM glycine pH 2.5. Bispecific complex mAb <tnt>rH-M-11-7-GthCol / / <igf1>-Fab-GthIM and mAb <tnt>rH-M-11-7-GthIM-IgG / / <igf1>The binding of TnT and IGF-1 to -Fab-GthCol was tested at 25°C. A series of experiments in which increasing concentrations of TnT (37 kDa) were injected in duplicate from 0.4 nM to 30 nM for 3.3 nM IGF-1 (7.7 kDa) and from 1.1 nM to 30 nM for 10 nM IGF-1 were performed at 30 μL / min, respectively, over the surface-displayed preformed bispecific complex. The association phase was monitored for 3 min, and the dissociation phase was monitored for 5 min. A schematic diagram is shown in Figure 17, and the results are shown in Figures 18-20 and Tables 4 and 5.

[0165] The GthCol and GthIM binding domains can be linked to a single IgG <tnt>- Two Fabs attached to the M-11-7 antibody <igf-1>This may facilitate the formation of bispecific antibody constructs composed of fragments. Different GthCol or GthIM antibody fusion constructs should elucidate the specificity and compatibility of antibody Gth binding domain combinations.

[0166] Fab <igf-1>-GthIM is surface-displayed IgG <tnt>-11-7-GthCol (B.) with affinity of K D = 40 pM. Molar ratio = 0.9 indicates a 1:1 binding stoichiometry. Fab <igf1>-GthCol and captured IgG <tnt>-No detectable binding with 11-7-GthCol (A.).

[0167] Fab <igf-1>-GthCol (C.) is surface-displayed IgG <tnt>-11-7-GthIM binds and forms a fast complex k a 4.8E+07 M -1 s -1 accompanied by dissociation k d 1.1E-03 s -1 results in a complex half-life t / 2-diss > 7 min. The affinity is K D = 35 pM. The molar ratio of 1.9 indicates a 2:1 binding stoichiometry. Fab <igf-1>-GthIM and captured IgG <tnt>-No detectable binding to 11-7-GthIM (D.). [Table 4]

[0168] The optimal combination for creating a bispecific antibody is C). Two Fabs <igf-1>-GthCol is IgG <tnt>-11-7-GthIM interacts with the two binding domains of GthIM, as indicated by a molar ratio of 1.9, similar to a 2:1 binding stoichiometry.

[0169] Next, antigen accessibility was tested. IGF-1 was found to be a 3.7E+06 M -1 s -1 and dissociation k d 1.4E-04 s -1 IgG <tnt>-11-7-GthCol / Fab <igf1>- binds to the GthIM complex, and the complex half-life time is t / 2-diss The resulting IGF-1 affinity was K D = 37 pM. A molar ratio of 1.4 indicates a 1:1 stoichiometry.

[0170] IGF-1 is a a 3.8E+06 M -1 s -1 and dissociation k d 8.1E-04 s -1 IgG <tnt>-11-7-GthIM / Fab <igf1>-GthCol complex, resulting in a complex half-life of t / 2-diss = 14 min with an affinity of K D = 212 pM. Molar ratio = 1.1 indicates a 1:1 binding stoichiometry.

[0171] TnT is k a 1.4E+06 M -1 s - and k d 1.4E-04 s -1 Bispecific complex IgG <tnt>-11-7-GthCol / Fab <igf1>-GthIM and exhibits a complex half-life of t / 2-diss 84 min. The affinity is D 97 pM. Molar ratio = 1.2 indicates a 1:1 stoichiometry.

[0172] TnT is k a 3.6E+06 M -1 s -1 and k d 1.4E-04 s -1( t / 2-diss 116 min) IgG <tnt>-11-7-GthIM / Fab <igf1>- binds to the GthCol complex, and K D = 28 pM and the molar ratio = 1.3 indicates a 1:1 stoichiometry. [Table 5]

[0173] The binding stoichiometry was calculated as follows: one TnT (37 kDa) molecule binds one IgG molecule with a 1:1 stoichiometry. <tnt>-11-7, one IGF-1 molecule (7.7 kDa) binds one Fab in a 1:1 stoichiometry <igf-1>This means that all IGF-1 binding sites displayed on the surface are saturated. <igf-1>Fragments are accessible. IgG <tnt>-11-7 binds to only one TNT antigen.

[0174] Example 3: Solid-phase immunoassay measurements Example 3.1: Elecsys ECL TnT measurement A commercially available cardiac TnT high-sensitivity Elecsys kit (Roche Diagnostics) GmbH, Elecsys Troponin T hs, Mat.050927744 190, Lot 42906601) and the biotinylated immunospecific factor in the kit was used as the antibody fragment Fab <tnt>The ruthenium-labeled immunospecific factor was the same as that used in the commercial kit. To capture the Fab fragments on paramagnetic beads, the biotinylated GthIM binding domain was immobilized on paramagnetic particles. This experiment was performed in triplicate on an Elecsys e411 instrument.

[0175] To determine the blank reference value, a Dilution Multi Assay (Material No. 03609987) was used. Troponin T hs CalSet (Material No. 0509275190) was used to determine TnT concentrations using Calibrator 1 containing 18 pg / ml TnT and Calibrator 2 containing 4200 pg / ml TnT. Biotinylated GthIM at 2 μg / ml in PBS and 10.3 μg / ml in PBS were used. <tnt>A new reagent was established in R1 using Fab-GthCol. As controls, 2 μg / ml GthIM in PBS and 10.3 μg / ml GthIM in PBS were used. <tnt>Fab-GthCol was used. Another control omitted the Fab fragment and used 2 μg / ml biotinylated GthIM in PBS.

[0176] Example 3.2: Prophetic example of Troponin T (TnT) measurement In another setting, isolation in an assay format that applies a solid phase or solid surface (e.g., beads, or multititer plate-based or Biacore formats) The detection of TnT in collected patient samples (e.g., serum or plasma) can be modified using a capture compound. This setup does not apply biotin and streptavidin. In this approach, the solid phase is directly coated with an immunoprotein, for example, via absorption or chemical coating known to those skilled in the art. The capture compound, i.e., an antibody or fragment thereof that specifically binds to troponin T, is covalently bound to a colicin (e.g., GthCol or ColE-7) so that the TnT-specific antibody can directly bind to the solid phase when exposed to the colicin-coated solid phase. The other steps, i.e., binding of the analyte to the capture compound and binding of the specificity factor conjugated to an indicator (detectable label), are carried out in a similar manner as described in the previous examples or as known to those skilled in the art for competitive assay formats.

[0177] In yet another embodiment, the capture compound, i.e., an antibody or fragment thereof that specifically binds to troponin T, is covalently bound to an immunity protein (e.g., GthIM or IM-7) so that when exposed to a colicin protein-coated solid phase, the TnT-specific antibody can bind directly to the solid phase. Subsequent steps can be carried out as described above.

[0178] References Backliwal et al.(2008)Biotechnology and Bioengineering 101:182 Cascales et al. (2007) Microbiol Mol Biol Rev 71(1):158 Doenecke et al. (1997), Leukemia 11:1787 Garinot-Schneider et al. (1996) J Mol Biol 260(5):731 Keeble et al. (2006) Biochemistry 45(10):3243 Kuhlmann et al. (2000) J Mol Biol 301:1163 Norderhaug et al. (1997), J. Immunol. Methods. 204:77 Wallis et al.1995 Biochemistry 34(42):13743 International Publication No. 2017 / 100584 A1 International Publication No. 2021 / 028309 A1 International Publication No. 2012 / 150321< / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt> < / tnt>

Claims

1. 1. A method for determining an analyte in a sample, comprising: (a) contacting the sample with (i) a binding compound that binds to the analyte, the binding compound comprising a binding agent and a first partner of an affinity pair (first affinity partner), and (ii) a second partner of the affinity pair (second affinity partner) bound to a solid surface, an indicator reagent, and / or a second binding agent; (b) determining the analyte based on the complex formed in step (a); and Including, A method in which one of the first affinity partner and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, and the other of the first affinity partner and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto.

2. 2. The method of claim 1 , wherein the binding agent comprises one of an antibody, an aptamer, anticalin, a designed ankyrin repeat protein, a receptor, or a fragment having binding activity to the analyte, and in one embodiment an antibody or a fragment thereof having binding activity to the analyte.

3. The method of claim 1 or 2, wherein the first affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, in one embodiment the polypeptide of SEQ ID NO: 13, and / or the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, in one embodiment the polypeptide of SEQ ID NO:

14.

4. 4. The method of any one of claims 1 to 3, wherein the sample is a sample from a subject, in one embodiment a tissue sample or a body fluid sample, and in a further embodiment a plasma, serum or blood sample.

5. 5. The method of any one of claims 1 to 4, wherein the binding compound is a capture compound and the second affinity partner is bound to a solid surface in step (b), in one embodiment the binding compound is a capture compound and the second affinity partner is bound to a solid surface and a further capture compound in step (b).

6. 6. The method of any one of claims 1 to 5, wherein the method further comprises contacting the sample with a detection compound, and wherein step (b) comprises determining the amount of complexes comprising the detection compound bound to the solid surface.

7. 6. The method of any one of claims 1 to 5, wherein the method further comprises contacting the sample with a characterization compound, and step (b) comprises determining the amount of complexes comprising the characterization compound bound to the solid surface.

8. 8. The method of any one of claims 1 to 7, wherein the binding compound comprises the amino acid sequence of SEQ ID NO: 10 or 12, and in one embodiment is covalently connected via at least one disulfide bridge to a polypeptide comprising, and in one embodiment consisting of, SEQ ID NO:

11.

9. A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or a sequence at least 85% identical thereto, wherein the amino acid at the position corresponding to position 77 of SEQ ID NO: 1 is not histidine, and in one embodiment is alanine, glycine, leucine, or isoleucine; and in one embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

16. Petite.

10. A polypeptide comprising the amino acid sequence specified in SEQ ID NO: 2 or a sequence at least 85% identical thereto, wherein (i) the amino acid at the position corresponding to position 17 of SEQ ID NO: 2 is not cysteine ​​and, in one embodiment, is alanine, serine, leucine, isoleucine, or glycine, and / or (ii) further comprises at least one functional peptide.

11. 11. The polypeptide of claim 9 or 10, wherein (i) the polypeptide further comprises a G residue immediately preceding the amino acid sequence, and in one embodiment, the amino acid sequence MG immediately preceding the amino acid sequence; (ii) the polypeptide further comprises a transglutaminase peptide, in one embodiment, a transglutaminase peptide comprising the amino acid sequence YRYRQ (SEQ ID NO: 15); (iii) the polypeptide further comprises a tag peptide, in one embodiment, a tag peptide comprising the amino acid sequence (His)6 (SEQ ID NO: 5), and in a further embodiment, a tag peptide comprising the amino acid sequence (His)8 (SEQ ID NO: 6); and / or (iv) the polypeptide further comprises at least one linker peptide.

12. A fusion polypeptide comprising the polypeptide of any one of claims 9 to 11 and a binding agent.

13. 10. A polypeptide complex comprising a first partner of an affinity pair (first affinity partner) and a second partner of the affinity pair (second affinity partner), wherein (i) the first affinity partner is the polypeptide described in claim 9 and the second affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a sequence at least 50% identical thereto, or (ii) the first affinity partner is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a sequence at least 50% identical thereto, and the second affinity partner is the polypeptide described in claim 10.

14. A polynucleotide encoding the polypeptide of any one of claims 9 to 11 and / or the fusion polypeptide of claim 12.

15. (I) a polypeptide (first affinity partner) according to claim 9 or a polynucleotide encoding the same, and / or (II) The polypeptide (second affinity partner) according to claim 10 or a polynucleotide encoding the same. Including, Housing included in the kit.

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